Systematic evaluation of characteristics of the membrane-based fed-batch shake flask

Biotech & Bioengineering

John

Scholl

et al. 2019

To overcome catabolite repression, industrial fermentation processes are usually operated in substrate-limited fed-batch mode. Therefore, the implementation of such an operating mode at small scale is crucial to maintain comparable process conditions. In this study, Bacillus licheniformis, a well-known producer of proteases, was cultivated with carbon (glucose)-and nitrogen (ammonium)-limited fed-batch conditions using the previously introduced membrane-based fed-batch shake flasks. A repression of protease production by glucose and ammonium was thus avoided and yields increased 1.5-and 2.1-fold relative to batch, respectively. An elevated feeding rate of glucose caused depletion of ammonium, which was recognizable within the oxygen transfer rate (OTR)signal measured with the Respiration Activity MOnitoring System (RAMOS). Ammonium limitation was prevented by feeding ammonium simultaneously with glucose. The OTR signal clearly indicated the initiation of the fed-batch phase and gave direct feedback on the nutrient release kinetics. Increased feeding rates of glucose and ammonium led to an elevated protease activity without affecting the protease yield (Y P/Glu ). In addition to Y P/Glu , protease yields were determined based on the metabolized amount of oxygenThe results showed that the protease production correlated with the amount of consumed glucose as well as with the amount of consumed oxygen. The membranebased fed-batch shake flask in combination with the RAMOS device is a powerful combination to investigate the effect of substrate-limited fed-batch conditions. K E Y W O R D SBacillus licheniformis, catabolite repression, fed-batch, membrane-based fed-batch shake flask, proteases

“…Thus, the concentration gradient between the culture broth (2.9 g/L) and the feed reservoir (0 g/L) causes

{NH}_{4}^{+}

to diffuse into the feed reservoir (Figure S1D). This phenomenon was also observed by Philip et al () for

{NH}_{4}^{+}

and

{PO}_{4}^{3 prefixnormal‐}

. From 45.5 hr onwards, the

{NH}_{4}^{+}

concentration in the feed reservoir and the culture broth approximate (Figure b).…”

Section: Resultssupporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Cultivation Conditionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Introducing substrate limitations to overcome catabolite repression in a protease producing Bacillus licheniformis strain using membrane‐based fed‐batch shake flasks

Biotech & Bioengineering

John

Scholl

et al. 2019

Establishing a Fed‐Batch Process for Protease Expression with Bacillus licheniformis in Polymer‐Based Controlled‐Release Microtiter Plates

Rauls

Egidi

et al. 2019

Biotechnology Journal

Introducing fed-batch mode in early stages of development projects is crucial for establishing comparable conditions to industrial fed-batch fermentation processes. Therefore, cost efficient and easy to use small-scale fed-batch systems that can be integrated into existing laboratory equipment and workflows are required. Recently, a novel polymer-based controlled-release fed-batch microtiter plate is described. In this work, the polymer-based controlled-release fed-batch microtiter plate is used to investigate fed-batch cultivations of a protease producing Bacillus licheniformis culture. Therefore, the oxygen transfer rate (OTR) is online-monitored within each well of the polymer-based controlled-release fed-batch microtiter plate using a µRAMOS device. Cultivations in five individual polymer-based controlled-release fed-batch microtiter plates of two production lots show good reproducibility with a mean coefficient of variation of 9.2%. Decreasing initial biomass concentrations prolongs batch phase while simultaneously postponing the fed-batch phase. The initial liquid filling volume affects the volumetric release rate, which is directly translated in different OTR levels of the fed-batch phase. An increasing initial osmotic pressure within the mineral medium decreases both glucose release and protease yield. With the volumetric glucose release rate as scale-up criterion, microtiter plate-and shake flask-based fed-batch cultivations are highly comparable. On basis of the small-scale fed-batch cultivations, a mechanistic model is established and validated. Model-based simulations coincide well with the experimentally acquired data.

Glucose‐containing polymer rings enable fed‐batch operation in microtiter plates with parallel online measurement of scattered light, fluorescence, dissolved oxygen tension, and pH

Biotech & Bioengineering

Czotscher

Klein

et al. 2019

Bioprocesses operated in batch mode can induce adverse effects like overflow metabolism, substrate inhibition, osmotic inhibition, oxygen limitation, and catabolite repression. To avoid these adverse effects, fed-batch is the predominant operation mode in industrial production. Nevertheless, screening for optimal production strains is usually performed in microtiter plates and shake flasks operated in batch mode without any online monitoring. Recently, a polymer-based controlled-release fedbatch microtiter plate with stable glucose release characteristics was described. In this study, a glucose-containing polymer matrix was used to manufacture polymer rings that were placed at the bottom of a 48-well microtiter plate. Thereby, the liquid content of the well became accessible for optical measurement by the BioLector device. Reflections caused by the polymer ring were minimized by adjusting the scattered-light measurement position. Influences on the measurement of the dissolved oxygen tension and pH could be avoided by choosing appropriate polymerring geometries. These adjustments enabled parallel online measurement of scattered light, fluorescence, dissolved oxygen tension, and pH of Escherichia coli BL21 (DE3) fed-batch cultivations. The online monitoring and fed-batch operation capabilities of the fed-batch microtiter plate presented in this study finds optimal application in screenings and initial process development.