When Do Two‐Stage Processes Outperform One‐Stage Processes?

Abstract

Apart from product yield and titer, volumetric productivity is a key performance indicator for many biotechnological processes. Due to the inherent trade-off between the production of biomass as catalyst and of the actual target product, yield and volumetric productivity cannot be optimized simultaneously. Therefore, in combination with genetic techniques for dynamic regulation of metabolic fluxes, two-stage fermentations (TSFs) with separated growth and production phase have recently gained much interest because of their potential to improve the productivity of bioprocesses while still allowing high product yields. However, despite some successful case studies, so far it has not been discussed and analyzed systematically whether or under which conditions a TSF guarantees superior productivity compared to one-stage fermentation (OSF). In this study, we use mathematical models to demonstrate that the volumetric productivity of a TSF is not automatically better than of a corresponding OSF. Our analysis reveals that the sharp decrease of the specific substrate uptake rate usually observed in (non-growth) production phases severely impacts the volumetric productivity and thus raises a big challenge for designing competitive TSF processes. We discuss possible approaches such as enforced ATP wasting to improve substrate utilization rates in the production phase by which TSF processes can become superior to OSF. We also analyze additional factors influencing the relative performance of OSF and TSF and show that OSF processes can be more appropriate if a high product yield is an economic constraint. In conclusion, a careful assessment of the trade-offs between substrate uptake rates, yields, and productivity is necessary when deciding for OSF vs. TSF processes.

Broadening the Scope of Enforced ATP Wasting as a Tool for Metabolic Engineering in Escherichia coli

“…The yield of the ATPase strain reached the maximum with 1.01 ± 0.08 product C‐atoms/glucose C‐atoms. In theory, the control strain should also reach the maximal possible yield but stayed with 0.92 ± 0.03 product C‐atoms/glucose C‐atoms somewhat below the maximum, which could be due to the increase of biomass (and thus a flux of carbon to the biomass) at the beginning of the cultivation as mentioned above. Because not all of the added glucose was consumed by the control strain, the product titer was 27.57% lower than in the ATPase strain (Figure B,F).…”

“…Generally, TSF processes separating growth and production may help to overcome the drawback of reduced volumetric productivity during OSF . In Klamt et al we hypothesized that ATP wasting in the production phase could further boost the performance of TSFs. We therefore analyzed the effect of overexpressed ATPase under growth‐arrested conditions (mimicking the production phase of a TSF) caused by nitrogen starvation, where the biomass of the ATPase strain and the control strain remain constant.…”

“…Moreover, the ATPase strain keeps these high rates—in contrast to the control strain—until all of the glucose is completely consumed. The resulting high volumetric productivity during stationary phase could help make TSF processes more efficient as envisioned in the theoretical study of our group . In contrast, if yield and titer are more important than productivity (e.g., for high‐value products), ATP wasting can be applied in a growth‐coupled mode in an OSF as described above.…”

“…One major aspect studied herein concerns the fact that due to the reduced formation of biomass (the catalyst of the bioconversion), the volumetric productivity (amount of product per time and volume) will likely decrease compared to the strain without ATP wasting . One approach to overcome such inherent trade‐offs between high product yield and high volumetric productivity is to use two‐stage fermentation (TSF) with decoupled growth and production phase in contrast to one‐stage fermentation (OSF) with growth‐coupled product synthesis as used in the aforementioned studies. In a recent theoretical study where the productivities of OSF and TSF processes were systematically compared, we found that enforced ATP wasting in the second (production) phase can significantly increase the productivity and thus the competitiveness of TSFs as it keeps a high driving force for substrate uptake also in the production phase where cell growth (usually consuming a large fraction of the substrate taken up) is missing .…”

Combinatorial impact of Sec signal peptides from Bacillus subtilis and bioprocess conditions on heterologous cutinase secretion by Corynebacterium glutamicum

“…Biomass specific product yield describes the efficiency of the biomass to synthesize the desired product from consumed substrate in bioprocesses with growth‐coupled product formation (Klamt, Mahadevan, & Hädicke, ; Looser et al, ). A high or low value of indicates a high or low usage of substrate for product synthesis in competition to biomass formation.…”