Application and Technical Notes
Anaerobic cultivation processes of probiotic bacteria in the BioLector XT microbioreactor
Scientific research on anaerobic or microaerophilic cultivation techniques, such as the cultivation of probiotics under microbiome-like conditions, is essential. Probiotics include a whole range of anaerobic bacteria such as Lactobacillus or Bifidobacterium.
We show the simplicity of using the BioLector XT’s microbioreactor gassing lid to perform anaerobic batch and fed-batch cultivations of the probiotic bacteria Lactobacillus casei, Lactobacillus plantarum and Bifidobacterium bifidum.
Using the BioLector XT Microbioreactor Gassing Lid
In this Technote we describe how to use the gassing lid, the different gassing options and all gassing lid variants configurable to your biological applications in the BioLector XT Microbioreactor and in the RoboLector XT Automated Microbioreactor.
Before the preparations of a physical cultivation setup, create a protocol in the BioLection software as described in the BioLector XT Microbioreactor user manual. Via the BioLection software you can choose between five different gassing modes. Depending on the biological application, a defined gassing option is essential for successful cultivation experiments, e.g., to realize a certain cultivation condition like dissolved oxygen in the cultivation medium
Screening yeast extract to improve biomass production in acetic acid bacteria starter culture
In this study, we demonstrate that the BioLector is a suitable device for online strain screening and for the investigation of the impact of different yeast extracts on biomass production of the acetic acid bacterium Komagataeibacter hansenii Ko- 0201. Experiments were performed under controlled and uncontrolled pH conditions.
We achieved improved biomass production of K. hansenii Ko-0201 in X-SEED® KAT by controlling pH. Furthermore, the pH-control in the low pH range between 4.5 and 5.5 was successful and a good correlation between online and offline values were achieved.
Adaptive Laboratory Evolution of Pseudomonas putida in the RoboLector
This application note demonstrates a successful ALE experiment for Pseudomonas putida performed with the RoboLector. It is shown that the RoboLector is a suitable cultivation system and an ideal tool for the easy performance of high-throughput ALE where time consuming steps like manual pipetting steps can be avoided. We validated the ALE application in the RoboLector by the successful reduction of the lag-phase determined by online colony screening.
Screening of yeast-based nutrients for E. coli-based recombinant protein production using the RoboLector
Currently, most particularly Escherichia coli, is used for recombinant protein production. E. coli has the advantage to grow rapidly on versatile substrates such as yeast hydrolysates and to yield high protein titers. Procelys, as one of the leading companies in yeast extracts (YE) and yeast peptones (YP) synthesis, provides high performance, animal free nutrients for biopharmaceutical manufacturing and more. As shown in this application note with the RoboLector platform only a few experiments are necessary to screen for the best media for protein production and induction time.
Determination of cell death in the BioLector II/ Pro
Extensive application examples using propidium iodide (PI) staining method in the BioLector Pro to identify dead or stressed cells during running cultivation processes are shown in the App Note. Online PI measurement during cultivation processes promises a rapid signal response, applicable e.g. in high throughput cytotoxicity screenings in 48 well format. In general, the PI staining is implemented to evaluate the cell viability of bacterial cultures from food products, clinical samples, environmental or fermentation processes or to characterize eukaryotic cells. There are still many development possibilities such as the calibration of the PI raw signal with the cell count of dead cells.
High throughput cultivation of the cellulolytic fungus Trichoderma reesei in the BioLector
In this application note we show, that the BioLector system is suitable for high throughput fermentation of T. reesei while maintaining essentially the same morphology, cellulase production and growth kinetics as in conventional shake flask fermentations. Furthermore, we demonstrate different application examples that take advantage of the optical online measurement capabilities of the BioLector. These examples include a reporter-based estimation of cellulase induction and online measurement of growth rates.
Low-pH profiling in μL-scale to optimize protein production in H. polymorpha using the BioLector Pro
In this application note a pH profiling for a maximized green fluorescent protein (GFP) yield in H. polymorpha has been conducted using the microbioreactor BioLector Pro. Within this high-throughput fermentation study, we investigated strains of H. polymorpha containing the FMD and the MOX promoter, to determine the preferred pH range for efficient protein production. Up to now, the investigated low pH range between pH 4 to 6 has been very challenging to measure and control in small scale cultivation systems.
Media optimization in the RoboLector Pro for enhanced protein production using C. glutamicum
In the research project described in this application note, we are presenting a media optimization of the minimal medium for Corynebacterium glutamicum (CGXII medium) using the RoboLector and BioLector Pro. The media optimization in the DoE is focused on increasing the cutinase activity in C. glutamicum. The organism is cultivated in batch and fed-batch processes using the BioLector Pro in order to investigate the influence of the media components and feeding rates on the growth-related secretory protein production of cutinase.
Feasibility study: Using the BioLector for Mammalian Cell Culture
Successful cultivation procedures with CHO DG44 2.14 8M and ExpiCHO-STM cells in the BioLector are presented in this application note. The results confirm the BioLector as a suitable device for the cultivation of mammalian cell cultures. In particular, online monitoring of biomass or fluorescence signals for cell growth, cell viability and the progress of protein expression in transfected cell cultures are detectable. All experimental data were kindly provided by Stefanie Schatz and Nicole Raven (Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen, Germany).
pH control settings in the BioLector Pro
Maintaining a stable pH value during the whole process is challenging and requires a rapid and precise pH control of the cultivation system. Therefore, an adjustable pH control for individual cultivation conditions is very essential. The BioLector Pro provides features to change the pH-control settings according to the requirements of the cultivation process. It combines the scalable BioLector technology with a microfluidic chip. The benefit of the BioLector Pro is the combination of high throughput batch or fed-batch cultivations in a micro titer plate (MTP) format with online non-invasive monitoring of biomass, fluorescence, DO and online control of pH.
E. coli fed-batch cultivation using the BioLector Pro
The BioLector Pro is ideally suited for microbial fed-batch cultivation processes. Up to 32 pH-controlled fed-batch cultivations can be performed in one experiment at the same time. This technical note gives an overall understanding of the use of the BioLector Pro on the example of a standard E. coli fed-batch cultivation experiment with one-sided pH-control. Standard experiment conditions are indicated and the standard protocol settings of the BioLection software are shown. For the fed-batch and pH-controlled cultivation experiments the microfluidic micro titer plate in the FlowerPlate format were used.
Cultivation of Suspended Plant Cells in the BioLector
Monitoring cultivations of suspended plant cells is often a laborious task. Especially because very few suitable online monitoring techniques are available in microliter scale. This gap is closed by using BioLector technology. The screening of cell lines or media optimization can be carried out conveniently, even along with design of experiments (DoE). Up to now, the BioLector was mostly used for the fermentation of bacteria or yeasts. In this application note, it is asserted that with the example of Nicotiana tabacum, that the BioLector technology can be utilized for suspended plant cell cultivation in microliter scale.
The BioLector's Scattered Light Signal
With the BioLector technology, it is not only possible to monitor biomass concentration online in microtiter plates in the form of scattered light, but also noninvasively and in a parallel manner in 48 wells. Conventionally, biomass analysis is performed offline after taking a sample, which is laborious and naturally confined to certain time points. In this application note, the principle of the BioLector's operation will be explained, and the factors that may influence experimental results. Furthermore, correlation to traditional biomass measurements is demonstrated, including the procedure of integrating this calibration function in the BioLection software.
Anaerobic High-Throughput Fermentations in the BioLector
Due to the volatility of the oil market, anaerobic bioprocesses are having a resurgence. Detailed process information on anaerobic fermentations is usually derived from standard stirred tank reactors, where an anaerobic atmosphere can be easily maintained and online monitoring data gained. Microtiter plates are already applied for the study of anaerobic organisms and processes in order to achieve high-throughput and lower cost and effort, but they lack the possibility of real-time measurement. In this application note, the adaption of the online monitoring BioLector technology to anaerobic requirements and its potential will be discussed.
RoboLector – Microbioreactor System for DO-Controlled Fed-Batch Cultivation
Bioprocesses ranging from bench to production scale are commonly operated in fed-batch mode. In contrast, up to now, performing fed-batch cultivations in microtiter plates has been difficult to realize. With the RoboLector system, it is now possible to perform up to 48 individually-controlled fed-batch cultivations at the same time and more. Based on all online recorded cultivation data within the BioLector, the liquid handling robot can carry out pre-defined manipulation actions, including sampling and the addition of liquids, like inducers or nutrients. This application note presents a detailed description of the computational control to realize DO-triggered fed-batch cultivations.
Mode of Operation of Optical Sensors for pH-Value and Dissolved Oxygen
The major objective for the use of microtiter plates in bioprocess development is how to gain significant process data for characterization and scale-up. The BioLector device enables customers to not only monitor biomass development in 48 parallel microbioreactors online, but the noninvasive and online detection of pH value and dissolved oxygen is also permitted via optical sensors (optodes). Optodes consist of special dyes that respond to environmental conditions. This technical note depicts the concept of the optodes' operational mode.
Guide for Cultivation in Microplates
For many compelling reasons, microplates have been used in laboratories for several decades. They are especially suitable if high throughput is needed. To use microplates as cultivation vessels, intense knowledge about the proper use is necessary, in order to be able to subsequently perform meaningful scale-ups. This guide provides an overview of the characteristics of different types of microplates available on the market.