Response of Corynebacterium glutamicum exposed to oscillating cultivation conditions in a two‐ and a novel three‐compartment scale‐down bioreactor

Abstract

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.

Metabolic profile of 1,5‐diaminopentane producing Corynebacterium glutamicum under scale‐down conditions: Blueprint for robustness to bioreactor inhomogeneities

“…Besides DAP as main product, C. glutamicum produces several side products such as amino-acids (Lemoine et al, 2015) and organic acids (Dominguez et al, 1993;Inui et al, 2004Inui et al, , 2007K€ aß et al, 2014b). Due to overflow metabolism in the initial batch phase in all processes, organic acids like L-lactate and succinate were produced in concentrations up to 0.3 mM (Llactate).…”

“…Although intermediary formation of acidic by-product has been identified in previous studies (K€ aß et al, 2014b;Lemoine et al, 2015;Limberg et al, 2016;Schilling et al, 1999), the basis of its metabolic robustness on molecular level is still not understood. Although intermediary formation of acidic by-product has been identified in previous studies (K€ aß et al, 2014b;Lemoine et al, 2015;Limberg et al, 2016;Schilling et al, 1999), the basis of its metabolic robustness on molecular level is still not understood.…”

Comparative performance of different scale‐down simulators of substrate gradients in Penicillium chrysogenum cultures: the need of a biological systems response analysis

“…() observed a 50% decrease in penicillin productivity as well as a higher turnover rate of storage pools relative to continuously fed chemostat cultivation (de Jonge et al ., , ). However, as recently argued (Haringa et al ., ), in these previous scale‐down studies, typically fluctuation timescales of 100–500 s have been applied (Vardar and Lilly, ; Neubauer and Junne, ; de Jonge et al ., ; Heins et al ., ; Lemoine et al ., ), which were based on the 95% mixing time () at industrial scales (Limberg et al ., ). However, a more realistic approach is to base the frequency of the substrate oscillations on the 4–5 times lower circulation time (Haringa et al ., ), which implies that the cells in reality experience faster changes in substrate concentration at timescales of tens of seconds, which has been recently confirmed from computational fluid dynamics (CFD) simulations of the 54 m 3 penicillin fermentation case (Haringa et al ., ).…”

Scale-up bioprocess development for production of the antibiotic valinomycin in Escherichia coli based on consistent fed-batch cultivations

“…As a high glucose concentration is connected to a high metabolic activity, also oxygen limitation appears in such a zone at the typical high cell concentrations [27]. In the case of production of small molecules such a feeding zone directly affects the flux through the central metabolic pathways [28] and thus may influence the yield of the product. To address this problem, a laboratory scale-down two-compartment reactor (TCR) was applied to simulate the effect of the feed zone and thus to study the robustness of the process [29].…”