Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates.

BACKGROUND: Substrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in industrial production processes. In contrast, screening processes are usually performed in microtiter plates operated in batch mode. This leads to a different physiological state of the production organism in early screening and can misguide the selection of potential production strains. To close the gap between screening and production conditions, new techniques to enable fed-batch mode in microtiter plates have been described. One of these systems is the ready-to-use and disposable polymer-based controlled-release fed-batch microtiter plate (fed-batch MTP). In this work, the fed-batch MTP was applied to establish a glucose-limited fed-batch screening procedure for industrially relevant protease producing Bacillus licheniformis strains. RESULTS: To achieve equal initial growth conditions for different clones with the fed-batch MTP, a two-step batch preculture procedure was developed. Based on this preculture procedure, the standard deviation of the protease activity of glucose-limited fed-batch main culture cultivations in the fed-batch MTP was ± 10%. The determination of the number of replicates revealed that a minimum of 6 parallel cultivations were necessary to identify clones with a statistically significant increased or decreased protease activity. The developed glucose-limited fed-batch screening procedure was applied to 13 industrially-relevant clones from two B. licheniformis strain lineages. It was found that 12 out of 13 clones (92%) were classified similarly as in a lab-scale fed-batch fermenter process operated under glucose-limited conditions. When the microtiter plate screening process was performed in batch mode, only 5 out of 13 clones (38%) were classified similarly as in the lab-scale fed-batch fermenter process. CONCLUSION: The glucose-limited fed-batch screening process outperformed the usual batch screening process in terms of the predictability of the clone performance under glucose-limited fed-batch fermenter conditions. These results highlight that the implementation of glucose-limited fed-batch conditions already in microtiter plate scale is crucial to increase the precision of identifying improved protease producing B. licheniformis strains. Hence, the fed-batch MTP represents an efficient high-throughput screening tool that aims at closing the gap between screening and production conditions.

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

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.

Validation of the transferability of membrane-based fed-batch shake flask cultivations to stirred-tank reactor using three different protease producing Bacillus strains.

Most industrial fermentation processes are operated in fed-batch mode to overcome catabolite repression, undesired by-product formation and oxygen limitation. To maintain comparable process conditions during screening of optimal production strains, the implementation of a fed-batch mode at small scale is crucial. In this study, three different protease producing Bacillus species, Bacillus aeolius, B. licheniformis and B. pumilus, were cultivated using the previously described membrane-based fed-batch shake flasks. Under carbon-limited conditions, catabolite repression was avoided, so that proteases were produced in all strains. Protease yields of B. aeolius and B. licheniformis increased 1.5-fold relative to batch cultivations. To validate process scalability between shake flasks and stirred tank reactors, membrane-based fed-batch shake flask cultivations were transferred to laboratory-scale stirred tank reactors with equal feeding rates. Despite inevitable differences between the scales such as pH control, feed supply and feed start, comparable results were achieved. Oxygen transfer rates of B. licheniformis and B. pumilus measured with the respiration activity monitoring system (RAMOS) in shake flasks and in stirred tank reactor with an off-gas analyzer were almost identical in both cultivation systems. The protease activities referring to the total consumed glucose were also mostly comparable. A slight decrease from shake flask to stirred tank reactor could be observed, which is presumably due to differences in pH control. This study successfully demonstrates the transferability of membrane-based fed-batch shake flask cultivations to laboratory-scale stirred tank reactors.

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

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 fed-batch 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 polymer-ring 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.

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

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 (YP/Glu ). In addition to YP/Glu , protease yields were determined based on the metabolized amount of oxygen (YP/O2) . The results showed that the protease production correlated with the amount of consumed glucose as well as with the amount of consumed oxygen. The membrane-based fed-batch shake flask in combination with the RAMOS device is a powerful combination to investigate the effect of substrate-limited fed-batch conditions.

High-throughput nucleoside phosphate monitoring in mammalian cell fed-batch cultivation using quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Current methods for monitoring multiple intracellular metabolite levels in parallel are limited in sample throughput capabilities and analyte selectivity. This article presents a novel high-throughput method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) for monitoring intracellular metabolite levels in fed-batch processes. The MALDI-TOF-MS method presented here is based on a new microarray sample target and allows the detection of nucleoside phosphates and various other metabolites using stable isotope labeled internal standards. With short sample preparation steps and thus high sample throughput capabilities, the method is suitable for monitoring mammalian cell cultures, such as antibody producing hybridoma cell lines in industrial environments. The method is capable of reducing the runtime of standard LC-UV methods to approximately 1 min per sample (including 10 technical replicates). Its performance is exemplarily demonstrated in an 8-day monitoring experiment of independently controlled fed-batches, containing an antibody producing mouse hybridoma cell culture. The monitoring profiles clearly confirmed differences between cultivation conditions. Hypothermia and hyperosmolarity were studied in four bioreactors, where hypothermia was found to have a positive effect on the longevity of the cell culture, whereas hyperosmolarity lead to an arrest of cell proliferation. The results are in good agreement with HPLC-UV cross validation experiments. Subsequent principal component analysis (PCA) clearly separates the different bioreactor conditions based on the measured mass spectral profiles. This method is not limited to any cell line and can be applied as a process analytical tool in biotechnological processes.