Microbioreactor Systems Research Articles

Optimization of a Bacterial Cultivation Medium via a Design-of-Experiment Approach in a Sartorius Ambr® 15 Fermentation Microbioreactor System

In the evolving landscape of sustainable biopharmaceutical process development, the utilization of bacteria in the production of various compounds via fermentation has attracted extensive attention from scientists. A successful fermentation process and the release of its associated products hinge on the synergy between an efficient bacterial strain and the formulation of a suitable growth medium. Balancing all nutrient levels of a growth medium to maximize microbial growth and the product quality is quite an intricate task. In this context, significant advancements have been achieved via the strategic implementation of design-of-experiment (DOE) methodologies and the utilization of parallel microbioreactor systems. This work presents a case study of the fermentation growth medium optimization of a Gram-negative bacterium of the Neisseriaceae family that releases outer membrane vesicles (OMVs), which represent a potential vaccine platform. To achieve this, the ability of Sartorius MODDE®13 DOE software to explore multiple variables and their interactions was combined with the functionality of a Sartorius Ambr® 15F parallel microbioreactor system. The findings reported in this study have led to the design of a well-suited fermentation medium for a Gram-negative bacterium and an improvement in the quality of the OMVs produced from it.

LED Illumination Modules Enable Automated Photoautotrophic Cultivation of Microalgae in Parallel Milliliter-Scale Stirred-Tank Bioreactors

Scalable lab-scale photobioreactors are needed for the exploration of new and improved photoautotrophic bioprocesses. Microbioreactor systems in which parallel bioreactors operate automatically are frequently employed to increase the speed of strain selection as well as the bioprocess-based exploration of heterotrophic fermentation processes. To enable the photoautotrophic operation of a commercially available parallel microbioreactor system with 48 stirred-tank bioreactors, LED illumination modules were designed to allow for individual light supply (400–700 nm) for each of the parallel bioreactors automated by a liquid handling station that performs both individual pH control and OD750 detection. The illumination modules enable dynamic variation of the incident light intensities of up to 1800 µmol m−2 s−1. Automated liquid level detection and volume control of each individual mL-scale gassed photobioreactor has to be established to compensate for evaporation because of the long process times of several days up to weeks. Photoautotrophic batch processes with Microchloropsis salina that employ either varying constant incident light intensities or day and night dynamics resulted in a standard deviation of OD750 of up to a maximum of 10%, with the exception of high-photoinhibiting incident light intensities. The established photoautotrophic microbioreactor system enables the automated investigation of microalgae processes in up to 48 parallel stirred photobioreactors and is thus a new tool that enables efficient characterization and development of photoautotrophic processes with microalgae.

Microbioreactor‐assisted cultivation workflows for time‐efficient phenotyping of protein producing Aspergillus niger in batch and fed‐batch mode

In recent years, many fungal genomes have become publicly available. In combination with novel gene editing tools, this allows for accelerated strain construction, making filamentous fungi even more interesting for the production of valuable products. However, besides their extraordinary production and secretion capacities, fungi most often exhibit challenging morphologies, which need to be screened for the best operational window. Thereby, combining genetic diversity with various environmental parameters results in a large parameter space, creating a strong demand for time-efficient phenotyping technologies. Microbioreactor systems, which have been well established for bacterial organisms, enable an increased cultivation throughput via parallelization and miniaturization, as well as enhanced process insight via non-invasive online monitoring. Nevertheless, only few reports about microtiter plate cultivation for filamentous fungi in general and even less with online monitoring exist in literature. Moreover, screening under batch conditions in microscale, when a fed-batch process is performed in large-scale might even lead to the wrong identification of optimized parameters. Therefore, in this study a novel workflow for Aspergillus niger was developed, allowing for up to 48 parallel microbioreactor cultivations in batch as well as fed-batch mode. This workflow was validated against lab-scale bioreactor cultivations to proof scalability. With the optimized cultivation protocol, three different micro-scale fed-batch strategies were tested to identify the best protein production conditions for intracellular model product GFP. Subsequently, the best feeding strategy was again validated in a lab-scale bioreactor.

Parallelized disruption of prokaryotic and eukaryotic cells via miniaturized and automated bead mill.

The application of integrated microbioreactor systems is rapidly becoming of more interest to accelerate strain characterization and bioprocess development. However, available high‐throughput screening capabilities are often limited to target extracellular compounds only. Consequently, there is a great demand for automated technologies allowing for miniaturized and parallel cell disruption providing access to intracellular measurements. In this study, a fully automated bead mill workflow was developed and validated for four different industrial platform organisms: Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Aspergillus niger. The workflow enables up to 48 parallel cell disruptions in microtiter plates and is applicable at‐line to running lab‐scale cultivations. The resulting cell extracts form the basis for quantitative omics studies where no rapid metabolic quenching is required (e.g., genomics and proteomics).

Parallelized microscale fed-batch cultivation in online-monitored microtiter plates: implications of media composition and feed strategies for process design and performance

Limited throughput represents a substantial drawback during bioprocess development. In recent years, several commercial microbioreactor systems have emerged featuring parallelized experimentation with optical monitoring. However, many devices remain limited to batch mode and do not represent the fed-batch strategy typically applied on an industrial scale. A workflow for 32-fold parallelized microscale cultivation of protein secreting Corynebacterium glutamicum in microtiter plates incorporating online monitoring, pH control and feeding was developed and validated. Critical interference of the essential media component protocatechuic acid with pH measurement was revealed, but was effectively resolved by 80% concentration reduction without affecting biological performance. Microfluidic pH control and feeding (pulsed, constant and exponential) were successfully implemented: Whereas pH control improved performance only slightly, feeding revealed a much higher optimization potential. Exponential feeding with µ = 0.1 h−1 resulted in the highest product titers. In contrast, other performance indicators such as biomass-specific or volumetric productivity resulted in different optimal feeding regimes.

A closer look at Aspergillus: online monitoring via scattered light enables reproducible phenotyping

BackgroundFilamentously growing microorganisms offer unique advantages for biotechnological processes, such as extraordinary secretion capacities, going along with multiple obstacles due to their complex morphology. However, limited experimental throughput in bioprocess development still hampers taking advantage of their full potential. Miniaturization and automation are powerful tools to accelerate bioprocess development, but so far the application of such technologies has mainly been focused on non-filamentous systems. During cultivation, filamentous fungi can undergo remarkable morphological changes, creating challenging cultivation conditions. Depending on the process and product, only one specific state of morphology may be advantageous to achieve e.g. optimal productivity or yield. Different approaches to control morphology have been investigated, such as microparticle enhanced cultivation. However, the addition of solid microparticles impedes the optical measurements typically used by microbioreactor systems and thus alternatives are needed.ResultsAspergillus giganteus IfGB 0902 was used as a model system to develop a time-efficient and robust workflow allowing microscale cultivation with increased throughput. The effect of microtiter plate geometry, shaking frequency and medium additives (talc and calcium chloride) on homogeneity of culture morphology as well as reproducibility were analyzed via online biomass measurement, microscopic imaging and cell dry weight. While addition of talc severely affected online measurements, 2% (w v−1) calcium chloride was successfully applied to obtain a highly reproducible growth behavior with homogenous morphology. Furthermore, the influence of small amounts of complex components was investigated for the applied model strain. By correlation to cell dry weight, it could be shown that optical measurements are a suitable signal for biomass concentration. However, each correlation is only applicable for a specific set of cultivation parameters. These optimized conditions were used in micro as well as lab-scale bioreactor cultivation in order to verify the reproducibility and scalability of the setup.ConclusionA robust workflow for A. giganteus was developed, allowing for reproducible microscale cultivation with online monitoring, where calcium chloride is an useful alternative to microparticle enhanced cultivation in order to control the morphology. Independent of the cultivation volume, comparable phenotypes were observed in microtiter plates and in lab-scale bioreactor.

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts, media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE's with a higher number of variables and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts, media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE's with a higher number of variables and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

Microbioreactor Systems for Accelerated Bioprocess Development.

In recent years, microbioreactor (MBR) systems have evolved towards versatile bioprocess engineering tools. They provide a unique solution to combine higher experimental throughput with extensive bioprocess monitoring and control, which is indispensable to develop economically and ecologically competitive bioproduction processes. MBR systems are based either on down-scaled stirred tank reactors or on advanced shaken microtiter plate cultivation devices. Importantly, MBR systems make use of optical measurements for non-invasive, online monitoring of important process variables like biomass concentration, dissolved oxygen, pH, and fluorescence. The application range of MBR systems can be further increased by integration into liquid handling robots, enabling automatization and, thus standardization, of various handling and operation procedures. Finally, the tight integration of quantitative strain phenotyping with bioprocess development under industrially relevant conditions greatly increases the probability of finding the right combination of producer strain and bioprocess control strategy. This review will discuss the current state of the art in the field of MBR systems and we can readily conclude that their importance for industrial biotechnology will further increase in the near future.

Integrated aeroelastic vibrator for fluid mixing in open microwells

Fluid mixing in micro-wells/chambers is required in a variety of biological and biochemical processes. However, mixing fluids of small volumes is usually difficult due to increased viscous effects. In this study, we propose a new method for mixing enhancement in microliter-scale open wells. A thin elastic diaphragm is used to seal the bottom of the mixing microwell, underneath which an air chamber connects an aeroelastic vibrator. Driven by an air flow, the vibrator produces self-excited vibrations and causes pressure oscillations in the air chamber. Then the elastic diaphragm is actuated to mix the fluids in the microwell. Two designs that respectively have one single well and 2 × 2 wells were prototyped. Testing results show that for liquids with a volume ranging from 10–60 µl and viscosity ranging from 1–5 cP, complete mixing can be obtained within 5–20 s. Furthermore, the device is operable with an air micropump, and hence facilitating the miniaturization and integration of lab-on-a-chip and microbioreactor systems.

A uniform-shear rate microfluidic bioreactor for real-time study of proplatelet formation and rapidly-released platelets.

Platelet transfusions, with profound clinical importance in blood clotting and wound healing, are entirely derived from human volunteer donors. Hospitals rely on a steady supply of donations, but these methods are limited by a 5-day shelf life, the potential risk of contamination, and differences in donor/recipient histocompatibility. These challenges invite the opportunity to generate platelets ex vivo. Although much progress has been made in generating large numbers of culture-derived megakaryocytes (Mks, the precursor cells to platelets), stimulating a high percentage of Mks to undergo platelet release remains a major challenge. Recent studies have demonstrated the utility of shear forces to enhance platelet release from cultured Mks. In this study, we performed a computational fluid dynamics (CFD) analysis of several published platelet microbioreactor systems, and used the results to develop a new 7-µm slit bioreactor-with well-defined flow patterns and uniform shear profiles. This uniform-shear-rate bioreactor (USRB-7µm) permits real-time visualization of the proplatelet (proPLT) formation process and the rapid-release of individual platelet-like-particles (PLPs), which has been observed in vivo, but not previously reported for platelet bioreactors. We showed that modulating shear forces and flow patterns had an immediate and significant impact on PLP generation. Surprisingly, using a single flow instead of dual flows led to an unexpected six-fold increase in PLP production. By identifying particularly effective operating conditions within a physiologically relevant environment, this USRB-7µm will be a useful tool for the study and analysis of proPLT/PLP formation that will further understanding of how to increase ex vivo platelet release. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1614-1629, 2017.

Framework for Kriging‐based iterative experimental analysis and design: Optimization of secretory protein production in Corynebacterium glutamicum

The production of bulk enzymes used in food industry or organic chemistry constitutes an important part of industrial biotechnology. The development of production processes for novel proteins comprises a variety of biological engineering and bioprocess reaction engineering factors. The combinatorial explosion of these factors can be effectively countered by combining high‐throughput experimentation with advanced algorithms for data analysis and experimental design. We present an experimental optimization strategy that merges three different techniques: (1) advanced microbioreactor systems, (2) lab automation, and (3) Kriging‐based experimental analysis and design. This strategy is demonstrated by maximizing product titer of secreted green fluorescent protein (GFP), synthesized by Corynebacterium glutamicum, through systematic variation of CgXII minimal medium composition. First, relevant design parameters are identified in an initial fractional factorial screening experiment. Then, the functional relationship between selected media components and protein titer is investigated more detailed in an iterative procedure. In each iteration, Kriging interpolations are used for formulating hypotheses and planning the next round of experiments. For the optimized medium composition, GFP product titer was more than doubled. Hence, Kriging‐based experimental analysis and design has been proven to be a powerful tool for efficient process optimization.

Characterization of oxygen transfer in a 24-well microbioreactor system and potential respirometric applications

The assessment of microbial processes is often done in Microbioreactor systems (MBRs), which allow for parallel cultivation in multiple independent wells. MBRs often include dissolved oxygen sensors, which are convenient for process characterization through oxygen uptake rate and other respirometric determinations. In order to assess respirometric potential of MBRs, a complete assessment of the DO fluorescent quenching sensors was done, showing that they presented a typical error of 0.56%, a signal to noise ratio of 189, a response time from 5.7 to 7.2s and no drift over a period of 24h. Then, KLa in the MBR was measured with different cassette and cap designs, liquid volumes, agitation rates, gas flow rates, temperatures and ionic strengths. KLa ranged from 8 to 90h−1, with a standard deviation between replicates from 2.8 to 17.5%. From these results and a numerical simulation, it was shown that the MBR tested allow the determination of oxygen uptake rates in a range from 0.038 to 3390mgL−1h−1, with a determination error less than 15%. Besides OUR determination, it was concluded that the MBR tested is also a convenient tool for dynamic pulse respirometry methods, based on experimental confirmation with four different cultures.

Automated microbioreactor systems for pharmaceutical bioprocessing: profiling of seeding and induction conditions in high-throughput fermentations

Introduction: Automated microbioreactor systems are designed for intensive bioprocess characterization. They facilitate reduction of development timelines without loss of valuable information. The RoboLector automated microbioreactor system was used for joint investigation of induction profiling and inoculation from seed cultures of different ages, which is only rarely recognized in literature for optimization. Results: The microbioreactor system allows reliable detection of growth phases and accurate inoculation procedures in combination with a true walk-away performance. Inocula taken from seed cultures resting in stationary growth phase for up to 10 h had no influence on induction profiling experiments, where late induction is preferred for maximum space-time-yield of recombinant enzyme production. Conclusion: The presented method allows for conduction of precise inoculation procedures and thus, for detailed studies on influential bioprocess parameters. The findings indicate that standardization in met...

Streamlined process development using the Micro24 Bioreactor system

The Pall Micro24 Bioreactor system is one of several microbioreactor systems that have been commercialised in recent years in response to the demand to reduce costs and shorten process development time lines. We have previously demonstrated that the Micro24 Bioreactor system can be integrated successfully into the later stages of cell line screening programmes and that the results correlate well with those from more conventional methods [1]. Further process development for these selected cell lines traditionally utilises bench top bioreactors to define appropriate process conditions giving the desired process outcomes although this approach can be time consuming and resource intensive. However the Micro24 Bioreactor system allows up to 24 different process conditions to be run concurrently thereby facilitating efficient process development. This work describes the use of the Micro24 Bioreactor system to identify improved process conditions for different cell lines and their subsequent validation in bioreactors.

Microfluidic bioreactor for dynamic regulation of early mesodermal commitment in human pluripotent stem cells

During development and regeneration, tissues emerge from coordinated sequences of stem cell renewal, specialization and assembly that are orchestrated by cascades of regulatory signals. The complex and dynamic in vivo milieu cannot be replicated using standard in vitro techniques. Microscale technologies now offer potential for conducting highly controllable and sophisticated experiments at biologically relevant scales, with real-time insights into cellular responses. We developed a microbioreactor providing time sequences of space-resolved gradients of multiple molecular factors in three-dimensional (3D) cell culture settings, along with a versatile, high-throughput operation and imaging compatibility. A single microbioreactor yields up to 120 data points, corresponding to 15 replicates of a gradient with 8 concentration levels. Embryoid bodies (EBs) obtained from human embryonic and induced pluripotent stem cells (hESC, hiPSC) were exposed to concentration gradients of Wnt3a, Activin A, BMP4 and their inhibitors, to get new insights into the early-stage fate specification and mesodermal lineage commitment. We were able to evaluate the initiation of mesodermal induction by measuring and correlating the gene expression profiles to the concentration gradients of mesoderm-inducing morphogens. We propose that the microbioreactor systems combining spatial and temporal gradients of molecular and physical factors to hESC and hiPSC cultures can form a basis for predictable in vitro models of development and disease.

Interview with Michel Maharbiz

Michel Maharbiz, an associate professor of Electrical Engineering and Computer Sciences (EECS) at UC Berkeley, haspushed his lab to the frontier of biological interface technologies and is increasingly interested in applications insynthetic biology. He received his B.S. in EECS from Cornell University in 1997 and his Ph.D. from UC Berkeley in 2003under Dr. Jay Keasling for his work on microbioreactor systems. Maharbiz’s lab has gained public recognition for theirwork on controlling the flight of insects by building so-called “cyborg beetles.” Though it is only one of many projectspursued by him and his co-investigators, it speaks clearly to his vision of a new era of technology: one that uses biointerfaceswith technology to harness nature’s complexity. MIT’s Technology Review named the cyborg beetle one ofthe top ten emerging technologies of 2009 and TIME magazine similarly hailed it as among the top fifty inventions thatsame year. Berkeley Scientific Journal met with Professor Maharbiz in 2011 to learn about his research and to explorehis ideas for the future of science.

Interview with Michel Maharbiz

Michel Maharbiz, an associate professor of Electrical Engineering and Computer Sciences (EECS) at UC Berkeley, has pushed his lab to the frontier of biological interface technologies and is increasingly interested in applications in synthetic biology. He received his B.S. in EECS from Cornell University in 1997 and his Ph.D. from UC Berkeley in 2003 under Dr. Jay Keasling for his work on microbioreactor systems. Maharbiz’s lab has gained public recognition for their work on controlling the flight of insects by building so-called “cyborg beetles.” Though it is only one of many projects pursued by him and his co-investigators, it speaks clearly to his vision of a new era of technology: one that uses biointerfaces with technology to harness nature’s complexity. MIT’s Technology Review named the cyborg beetle one of the top ten emerging technologies of 2009 and TIME magazine similarly hailed it as among the top fifty inventions that same year. Berkeley Scientific Journal met with Professor Maharbiz in 2011 to learn about his research and to explore his ideas for the future of science.