The oscillatory conditions in substrate and oxygen supply that typically occur on a large (industrial) scale are usually simulated in two-compartment scale-down reactors. In this study, the performance of nutrient-limited fed-batch cultivations of Corynebacterium glutamicum in a standard two-compartment reactor (two-CR) is compared to the performance in a novel three-compartment reactor (three-CR). The three-CR is designed to mimic three distinct zones of an industrial scale bioreactor that occur if the feed addition is installed at the bottom of the fluid phase. Our findings show that lactate and succinate appear in concentrations two-fold higher in the three-CR cultivation than in the two-CR cultivation. Similar results are revealed for the amino acids glycine, threonine, glutamate, and glutamine. In contrast to the two-CR cultivation, no intracellular accumulation of pyruvate is observed in the three-CR cultivation, since the carbon fluxes are directed toward lactate. As previously reported, the expression of lactate dehydrogenase (LDH) is increased in the context of oxygen deprivation. Thus, C. glutamicum adapts to the oscillating environment in the three-CR. This successful adaptation is revealed by a flow cytometric analysis of BOX-stained cells and a series of electrooptical at line measurements of cell polarisability. Both methods indicate a higher polarisability of cells in the three-CR cultivation. PI-staining does not indicate any membrane damage or accelerated cell death in either system. However, although the strain shows robustness, the product yield of lysine is reduced in scale-down cultivations as compared to cultivations at homogeneous conditions, which underlines the relevance of process optimization.

Performance loss of Corynebacterium glutamicum cultivations under scale-down conditions using complex mediaSubstrate and oxygen gradients appear in industrial-scale fed-batch processes due to limitations in the achievable power input and concomitantly increased mixing times. In order to mimic these gradients at lab scale, scale-down reactors are applied. Previous studies in such reactor systems suggest that Corynebacterium glutamicum is robust against oscillatory oxygen and substrate availability in relation to growth and side product accumulation. Usually, defined mineral salt media are applied contrary to the industrial case, in which complex media containing different carbon sources are used. Therefore, this study investigated the cultivation performance using complex medium based on sucrose, molasses, and corn steep liquor in a threecompartment scale-down reactor. The reactor consisted of a stirred tank and two plug flow reactor modules. This approach was applied based on assumptions of gradient distributions in bottom-fed bioreactors. A drastic reduction of growth and volumetric product yield of a cadaverine producing strain was observed while several short chain fatty acids accumulated, among them l-lactate and acetate. Growth was depleted after several hours of cultivation, while the substrate uptake rate was reduced by 20%. Hence, the main carbon source sucrose accumulated after 10 h of fed-batch cultivation. Despite growth cessation, neither reduction of cell vitality nor increased cell lysis were observed. IntroductionAlthough progress has been made in the field of parallel automated bioprocess development, which includes several on-, at-, and offline analyses and model-based strategies to identify the best strain and cultivation conditions, scale-up of a bioprocess

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The growth of the world's population, predicted to reach 9.3 billion by 2050, along with resource limitation, soil degradation and other threats to food security, calls for innovative strategies for food production and consumption. Currently, a shift from meat to fish intake is encouraged to improve sustainability and mitigate the impact of food systems, particularly in low-income rural areas (van der Goot

Corynebacterium glutamicum is well-known as an industrial workhorse, most notably for its use in the bulk production of amino acids in the feed and food sector. Previous studies of the effect of gradients in scale-down reactors with complex media disclosed an accumulation of several carboxylic acids and a parallel decrease of growth and product accumulation. This study, therefore, addresses the impact of carboxylic acids, for example, acetate and L-lactate, on the cultivation of the cadaverine producing strain C. glutamicum DM1945Δact3:P tuf -ldcC opt and their potential role in scale up related performance losses. A fluctuating power input in shake flask and stirred tank cultivations with mineral salt was applied to mimic discontinuous oxygen availability.Results demonstrate, whenever sufficient oxygen was available, C. glutamicum recovered from previously occurring stressful conditions like an oxygen limiting phase.Reassimilation of acids was detected simultaneously. In cultures, which were supplemented with either acetate or L-lactate, a rapid cometabolization of both acids in presence of glucose was observed, showing conversion rates of 7.8 and 3.8 mmol g cell dry weight −1 hr −1 , respectively. Uptake of these acids was accompanied by increased oxygen consumption. Proteins related to oxidative stress response, glycogen synthesis, and the main carbon metabolism were found in altered concentrations under oscillatory cultivation conditions. (Proteomics data are available via ProteomeXchange with identifier PXD012760). Virtually no impact on growth or product formation was observed. We conclude that the reduced growth and product formation in scale-down cultivations when complex media was used is not caused by the accumulation of carboxylic acids. K E Y W O R D S carboxylic acid, Corynebacterium glutamicum, heterogeneity, oxygen oscillation, scale down