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Bacterial Fermentation
Bioreactors for bacteria
Fermentation process development
Fermentation scale-up and scale-down
Eppendorf bioreactors for bacterial fermentation
Fermentation of industry-relevant bacteria
Customer success stories
What is bacterial fermentation?
Bacterial fermentation is a metabolic process in which bacteria break down carbohydrates and amino acids to obtain energy for growth. Industrial fermentation is the process of using microbes to produce plasmids, enzymes, proteins, peptides, and other products on a large scale. Due to this, bacterial fermentation is used in many industrial settings including the manufacture of biopharmaceuticals, food and feed, chemicals, and biofuels. In the biopharmaceutical industry, microorganisms are a tool to produce therapeutics such as plasmids, proteins, peptides, and small molecules. Here, we will mainly talk about the applications of bacteria like Escherichia coli, and how to optimize E. coli production and other bacterial fermentations. However, bacteria are not the only microorganisms used to produce chemicals and therapeutics. Find out more about yeast fermentation and its applications in biopharma.
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Bioreactors for bacteria
What are the benefits of bioreactors for bacterial fermentation?
The benefits of bioreactors for bacterial fermentation compared to shake flasks include
- Better control of critical process parameters to create optimal growth conditions
- Achievement of higher cell densities through automated culture feeding
- Better scalability through scalable bioreactor design
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Customer case study
Are you interested in an example of the benefits of bioreactors for bacterial fermentation?
Then have a look at this customer case study about protein production in E. coli.
It shows that:
- Shake flasks were useful at the beginning of the optimization process to optimize the medium composition and induction conditions.
- Switching from shaker to bioreactor and the implementation of a fed-batch feeding strategy led to a 27-fold increased cell concentration.
What to consider when choosing a bioreactor for bacterial fermentation
Strain selection, choice of media, process development and scale-up are all key to maximizing the productivity of your bacterial fermentation process. Suitable bioreactors need to be able to facilitate these procedures to produce high quality and optimal yield of the desired products.
To achieve high cell densities and product yields in bacterial fermentation, it is essential that the bioreactor system enables the following:
Read on in the sections below for more information on these parameters.
To achieve high cell densities and product yields in bacterial fermentation, it is essential that the bioreactor system enables the following:
- Tailored gas supply for aerobic, microaerophilic, and anaerobic fermentation
- Nutrient supply via culture feeding
- Temperature regulation
- Culture scalability
Read on in the sections below for more information on these parameters.
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What to consider to tailor oxygen supply in bacterial fermentation
Aerobic fermentation
The necessary oxygen level for cultures of aerobic bacteria and yeast is typically 10 % to 50 % of air saturation. Keeping DO levels above the threshold amount by ensuring there is a suitable supply of oxygen is critical to sustaining optimal development. There are several bioreactor components that influence this:
- Impellers: These are used to agitate and mix a cell culture. The number of blades, speed and diameter can all affect the DO level.
- Sparger: Spargers are tools for gas exchange, specifically for introducing air and oxygen. The type of sparger influences the created bubble size and how effectively oxygen it is dispersed in the medium and therefore how easy is it made available to the cell biomass.
- Gassing system: Gasses can be introduced into the bioreactor via the headspace or directly into the culture. This is called overlay and submerged gassing, respectively.
- Baffles: The use of baffles enhances the mixing process. It leads to more turbulent mixing and prevents the culture from vortex mixing by breaking the vortex.
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OTR and kLa
The bioreactor’s oxygen transfer rate (OTR) and the volumetric oxygen transfer coefficient (kLa) are used to describe the oxygen transfer to the culture medium in the bioreactor. They are influenced by impellers, spargers, gassing systems and baffles, among other factors. Determining the OTR and kLa enables you to optimize the bioprocess in bioreactors and ensures a successful scale-up procedure. Click here to read a protocol for OTR determination and a protocol for kLa determination of bioreactors, both presented by the Eppendorf bioprocess application team.
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Anaerobic fermentation
This describes an oxygen-free environment or a DO concentration of 0 %, and is commonly used in microbial production to manufacture various compounds including ethanol, lactic acid, and acetic acid.
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Anaerobic fermentation application example
As an example for anaerobic fermentation in bioreactors, download this application note about anaerobic Clostridium beijerinckii fermentation for solvent production.
It shows that:
- Anaerobic fermentation is feasible in DASbox® Mini Bioreactor System and DASGIP® Parallel Bioreactor Systems equipped with BioBLU® f Single-Use Bioreactors
- Control of the redox potential led to a 6-fold increase in bacterial butanol production
- The scale-up strategy based on constant tip speed resulted in comparable redox and pH trends, as well as comparable bacterial growth, and butanol production
Micro-aerophilic fermentation
Conditions of less than 10 % DO are usually required for micro-aerobes, such as those found in the human gut. Probiotics are frequently produced using this type of fermentation. Culturing microbiome bacteria requires specific growth conditions that mimic the bacteria’s natural environment such as the gut or soil. This generally requires fine control of key parameters such as oxygen, pH, temperature and substrates. For example, in order to cultivate Lactobacilli and their natural fermentation by-products, bioreactors need to be able to reproduce the microaerobic conditions found in the gastrointestinal tract.
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Micro-aerophilic fermentation application example
Download this application note to learn how to replicate the microaerophilic conditions of the gut using a bioreactor.
It shows that:
- Precise control of dissolved oxygen at 4 % is feasible using both the BioFlo® 120 and BioFlo® 320 bioprocess controllers. A DO level of 4 % is equivalent to an oxygen concentration of 0.8 %, representing the physiological conditions of the gut microbiome.
- Both the BioFlo 120 and BioFlo 320 bioprocess controller facilitated robust growth of Lactobacillus acidophilus.
What to consider to supply nutrients via culture feeding in bacterial fermentation
Bacteria require nutrients to grow. In a bioprocess, nutrients need to be supplied by culture feeding and the feeding strategy will influence bacterial growth. Here are three different feeding strategies for bioreactor cultures:
Fed-batch and continuous culture operation modes offer the possibility to incorporate feed automation and improve your fermentation process. Feed automation can be accomplished by tracking various parameters in real-time to keep track of the culture's metabolic health and automatically alter the substrate concentration with feed pumps. Bioprocess software like DASware® control can be utilized to automate feeding by monitoring important parameters in real-time.
- Batch: Feeding substrate is only added at the beginning of the fermentation process.
- Fed-batch: Feeding substrate is added at the beginning and in increments throughout the rest of the bioprocess until the maximum working volume is reached.
- Continuous: Substrate is continuously added to the culture, with the continuous harvesting of cells and media to maintain a constant environment.
Fed-batch and continuous culture operation modes offer the possibility to incorporate feed automation and improve your fermentation process. Feed automation can be accomplished by tracking various parameters in real-time to keep track of the culture's metabolic health and automatically alter the substrate concentration with feed pumps. Bioprocess software like DASware® control can be utilized to automate feeding by monitoring important parameters in real-time.
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What to consider to regulate the temperature in bacterial fermentation
Microorganisms have optimal temperature ranges where enzymes can function properly. Since cell growth and metabolic function are heavily dependent on certain enzymatic pathways, microbial production will be significantly impacted if temperatures rise or fall outside of these ranges. It’s therefore essential that during the bioprocess the temperature is monitored and adjusted when necessary. Due to the high metabolic activity of many microbes, overheating can be a much more serious problem than under-heating for a microbial culture so it’s important to have components that can help to cool a microbial culture.
Ways to cool a microbial culture to maintain a stable temperature in a bioreactor:
Ways to cool a microbial culture to maintain a stable temperature in a bioreactor:
- Use of stainless-steel cooling fingers
- Use of water-jacketed vessels
- Some variants of the BioBLU® f Single-Use Βioreactor product line have patented cooling baffles, providing efficient heat removal through active cooling of the interior of the bioreactor.
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What to consider when scaling up bacterial cultures
Bioprocess development is initially conducted using small volumes until the ideal conditions for microbial production have been found. A scaling up process is then carried out to reach the larger volumes required for production. This procedure is generally carried out in stages, from bench scale to pilot scale through to industrial scale. An important goal when scaling-up is to reproduce process performance across scales.
Visit our bioprocessing scale-up webpage to learn more about bioprocess scale up strategies and the Eppendorf bioprocess solutions for bioprocessing scale-up.
Visit our bioprocessing scale-up webpage to learn more about bioprocess scale up strategies and the Eppendorf bioprocess solutions for bioprocessing scale-up.
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Bacterial fermentation process development
Getting started with bacterial cultivation in fermentors
Read on for some advice on how to start simple with bacterial fermentation in bioreactors!
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Use an easy-to-use bioprocess system
One simple starting point is an easy-to-use bioprocess control system. The BioFlo® 120 , for example, is a user-friendly bioprocess controller for bacterial fermentation thanks to its Auto Culture Mode software feature. This feature allows beginners to achieve satisfactory results in a short time and provides a basis for further optimization. The Auto Culture Mode is populated with setpoints and cascades that have been tested by our applications development team. Users only need to select the vessel size and type from the available options, and make standard preparations (sensor and pump calibration, vessel preparation) for the run.
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Application example: Auto Culture Mode
To find out more about the BioFlo® 120 Auto Culture Mode, read this application note from our bioprocess application experts.
Application note highlights:
- The application note explains E. coli Auto Culture mode setpoints and loop modes.
- The Auto Culture Mode reduced the complexity of the design of a new E. coli fermentation bioprocess.
- Culturing E. coli in Auto Culture Mode resulted in a successful batch run.
Benefit from how-to-guides for bacterial fermentation
If you are just getting started with E. coli culture in bioreactors, you might look for information on how to set up an E. coli bioprocess. Our application experts have developed guides, protocols, and information that help you getting started. Find some recommended readings below.
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Protocol: E. coli bioprocess setup at small scale.
Download this protocol on E. coli bioprocess setup using the DASbox® Mini Bioreactor System or DASGIP® Parallel Bioreactor System .
- The protocol explains how to prepare and conduct E. coli fermentation processes in the DASbox Mini Bioreactor System and the DASGIP Parallel Bioreactor System
- The protocol explains how bioprocess workflow steps differ with the use of glass and single-use bioreactors
- The protocol explains which bioprocess parameters and control strategies can be optimized and can serve as a starting point for further optimization
Application Note: A beginners guide to bioprocess modes
Discover a beginner's guide to bioprocess modes, authored by our bioprocess application experts.
Summary:
- The application note explains the differences between batch, fed-batch, and continuous fermentation and how these influence bacterial growth
- The application note explains methods to determine biomass, bacterial growth rate, productivity, yield, and analysis of process costs
- The application note explains the preparation of bacterial culture medium, inoculum, vessel setup, sensor calibration, and bioprocess run
Bioprocess expert tips
Discover the top 10 bioprocess expert tips from our applications lab:
- Tips to simplify the handling of your bioreactor system and vessels
- Tips to save time in sensor calibration
- Tips to improve dip tube cleaning, avoid filter clogging, and more
How can I accelerate upstream process development in bacterial fermentation?
To establish a microbial production process that is robust and efficient, scientists need to understand how critical process parameters influence each other and the resulting product. Process optimization can be simplified and accelerated by
- Parallel bioreactor systems: These systems enable the simultaneous operation of multiple experiments, thereby conserving time and resources while using lab space efficiently. One example of a parallel bioreactor system is the DASbox® Mini Bioreactor System .
- Single-use bioreactors: They eliminate the need for labor-intensive cleaning, improve turn-around time, reduce the cross contamination risk, and consequently, increase the effectiveness of your microbial production. BioBLU® f Single-Use Bioreactors are an example of single-use bioreactors for microbial applications.
- Bioprocess control software enables critical process parameters such as pH, temperature, dissolved oxygen, nutrient concentration and the organism's status to be measured and controlled at setpoints. Click here for more information about how bioprocess monitoring and control works.
- Software tools to turn data into knowledge. In-depth data analysis is required to understand how process parameters influence each other and the product. BioNsight® cloud is one example of a software tool that can advance bioprocess data analysis.
- Bioprocess automation: The development and execution of fermentation processes in bioreactors can be simplified and accelerated by process automation. Examples of automatable process steps in bioprocessing include bioprocess sampling , process parameter control, culture feeding, and data handling. Click here for more information about automating routine tasks in bioprocessing.
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How can single-use fermentors help save process development time?
Single-use bioreactors can help save time in bacterial fermentation by minimizing setup times, eliminating cleaning procedures and reducing labor time and costs.
BioBLU® f Single-Use Bioreactors are a powerful tool for more efficient process development:
- They achieve the mass transfer and heat removal requirements of fermentation processes by
- Proven stirred-tank design
- Powerful overhead drives featuring Rushton-type impellers
- Smart solutions for cooling
Application example: BioBLU® f Single-Use Bioreactors
Read this application note to see how BioBLU® f Single-Use Bioreactors suit the needs of E. coli fermentation.
It shows that:
It shows that:
- OTR values measured in the BioBLU® 0.3f Single-Use Bioreactors were comparable to those achieved with conventional DASbox® glass bioreactors
- Final biomass achieved in BioBLU® Single-Use Bioreactors and glass bioreactors were comparable
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How can I automate sampling in bacterial fermentation to reduce manual work?
In bioprocessing, sampling can be automated using an autosampler. Automated sampling facilitates sampling 24/7 in regular short intervals and frees up time for other tasks.
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Application example: Autosampling
Find out more about automated sampling of an E. coli bioprocess in an application note developed by our bioprocess applications team.
Application note highlights:
- The Bioprocess Autosampler enabled the efficient monitoring and evaluation of feeding strategies by automating the sampling process.
- Using the Bioprocess Autosampler enabled sampling of 200 samples from an 8-fold parallel DASbox Mini Bioreactor System over the course of 48 hours.
- The Bioprocess Autosampler enabled sampling outside working hours. Consistent sampling intervals enabled conclusions to be drawn about the preferred feeding strategy, which would have been overlooked if sampling had only occurred during normal working hours.
- Optical density and glucose concentration remained stable in samples stored in the autosampler's cooling stack at 4 °C for 72 hours.
How can I automate culture feeding in bacterial fermentation?
Culture feeding impacts bioprocess productivity, product quality, as well as the bioengineer’s workload. Its optimization is therefore a central part of upstream bioprocess development. Advantages of feed automation in bacterial fermentation include:
To automate feeding, two steps need to be taken:
- Prevention of nutrient depletion
- Generation of a more stable macro-environment
- Reaching higher product yields
- Reduction of manual workload
- Improvment of standardization
To automate feeding, two steps need to be taken:
- First, a feed pump needs to be installed, that can be controlled by the bioprocess control software and can add the feed solution into the bioreactor automatically,
- Second, the bioprocess control software must be programmed to activate and control the pump.
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Strategies for feed automation in bioprocessing
- Time-based feeding: A defined volume of feed solution is added to the culture per time increment. One example is an exponential feed profile. Exponential feeding of an actively growing culture aims at elongating the exponential growth phase for higher biomass density, while effectively avoiding nutrient depletion and toxic byproduct buildup.
- Feeding based on a DO spike: The DO concentration in a bioreactor depends on both air supply and cellular oxygen consumption, decreasing as cells grow. When carbon sources are exhausted, cell metabolism drops, causing a sudden DO spike. This spike signals substrate depletion and can be used to automate culture feeding. Feeding based on a DO spike is particularly suited for microbial cultures with a high oxygen demand.
- Feeding based on substrate concentration: The concentration of a relevant substrate (e.g. glucose) is directly measured. If it drops below a threshold, feeding is initiated. Feeding based on the substrate concentration allows a particularly close control of one specific nutrient, in most cases the major carbon source.
- Feeding based on the respiratory quotient: The respiratory quotient is the quotient of carbon dioxide produced and oxygen consumed by a culture. A drop of the RQ below 1 indicates glucose depletion and triggers the initiation of a feed pump.
If you are interested in a more detailed explanation of the different feeding strategies and how to implement them using software scripts, download our ebook: Upstream bioprocessing – Improving efficiency through digital tools .
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Application example: Feed automation
Read this application note by our customer at TU Wien to find out how a biomass soft sensor was used to control various substrate uptake rates.
It shows that:
- A biomass soft sensor was implemented using a DASbox® Mini Bioreactor System and DASware® control software .
- Biomass of the E. coli culture was estimated online using the soft sensor and based on the data the pump flow rate was automatically adjusted to control the supply of culture feed.
How can I optimize plasmid production in a bioreactor?
Plasmids are circular molecules of DNA that are found naturally occurring in bacteria and can replicate independently of genomic DNA. Plasmid DNA serves an essential role in bioprocessing - particularly for biopharmaceutical applications – as it is involved in the production of recombinant proteins and viral vectors.
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Application note: Plasmid production in bioreactors
Find more information about plasmid production in bioreactors from our application experts. Download this application note about E. coli plasmid DNA production using the DASbox® Mini Bioreactor System .
It shows that:
- Plasmid DNA production in the DASbox® Mini Bioreactor System was feasible.
- Material and methods for plasmid DNA production in bioreactors are described.
- In a fed-batch process the optical density as well as plasmid yield were higher than in a batch process.
- Plasmid production yield and cell growth correlated in both the batch and the fed-batch bioprocess.
Bacterial fermentation scale-up and scale-down
How can I scale-up or scale-down bacterial fermentation in bioreactors?
Various scaling strategies can be used to scale-up or scale-down bacterial fermentation in bioreactors.They often aim at keeping one process parameter constant across scales, such as power input per liquid volume, tip speed, kLa, or the mixing time.
Visit our bioprocessing scale-up page to find more information about different scale-up strategies and about the scalable fermentor solutions from Eppendorf.
Visit our bioprocessing scale-up page to find more information about different scale-up strategies and about the scalable fermentor solutions from Eppendorf.
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Application note: Scalability of E. coli fermentation in single-use fermentors
This application note analyses the scalability of bacterial fermentation in BioBLU® f Single-Use Bioreactors .
Summary:
- E. coli bioprocesses were carried out in BioBLU® 0.3f and BioBLU® 1f Single-Use Bioreactors with starting working volumes of 100 mL and 700 mL, respectively.
- Fermentation runs in BioBLU 0.3f and BioBLU 1f Single-Use Bioreactors resulted in comparable cell wet weights.
Where can I find examples of bacterial fermentation scale-down?
Download the following application notes for some examples for successful process scale-down using Eppendorf bioreactor systems.
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Application note: Scale-down of a C. glutamicum fermentation process
Download this customer application note about fermentation scale-down.
Summary:
- Scientist at Forschunszentrum Jülich used a DASGIP® Parallel Bioreactor System and DASware® control software to set up a scale-down model to simulate inhomogeneous cultivation conditions as they can occur at production scale
- They were able to simulate parameter gradients that can occur at production scale and evaluated metabolic effects of the oscillating DO and substrate concentrations.
Application note: Scaling down E. coli fermentation
This customer application note describes the scale-down of an E. coli fermentation process for amino acid production.
Application note highlights:
- Scientists at Evonic used a DASbox DASbox® Mini Bioreactor System Mini Bioreactor System to scale-down a 2 L bioprocess to 200 mL.
- Critical process parameters such as feeding profiles, impeller tip speed, and pH, DO, and temperature setpoints were successfully transferred from a benchtop bioreactor system to the DASbox system.
- Product yields in the DASbox system were comparable to those in the benchtop bioreactor system
Eppendorf bioreactors for bacterial fermentation
Eppendorf offers bioprocess controllers , scalable glass and single-use bioreactors , software solutions , and services to help accelerate your upstream bioprocess development, automate routine tasks, maximize the use of your bioprocess data, and tranfer your process from R&D to manufacturing .
Please see below for some of our bioprocess solutions for bacterial fermentation at different scales. Our bioprocess specialists are happy to provide further support. Please contact us to discuss the needs of your individual application.
Please see below for some of our bioprocess solutions for bacterial fermentation at different scales. Our bioprocess specialists are happy to provide further support. Please contact us to discuss the needs of your individual application.
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A selection of Eppendorf bioreactors for bacterial fermentation
Cultivation of industry-relevant bacteria in Eppendorf bioreactors
Corynebacterium glutamicum
Corynebacterium glutamicum is an industrial microbe traditionally used to produce amino acids for the food and feed industries. Advances in our genetic understanding and capabilities have enabled the creation of a versatile and effective platform to produce recombinant proteins and small molecules.
Download the following application notes that demonstrate how our customers successfully cultivated C. glutamicum in Eppendorf bioreactor systems!
Download the following application notes that demonstrate how our customers successfully cultivated C. glutamicum in Eppendorf bioreactor systems!
- Application Note 304: A DASGIP® Parallel Bioreactor System was used to produce chemicals from engineered C. glutamicum strains.
- Application Note 301: A DASGIP® Parallel Bioreactor System and DASware® control software were used used for scale-down model development.
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Streptococcus sp.
Streptococcus fermentation is one method used to manufacture hyaluronic acid (HA), a key molecule in a variety of medical, pharmaceutical, nutritional and cosmetic applications.
Download the following application note that demonstrates how one of our customers successfully cultivated Streptococcus sp. in Eppendorf a bioreactor system:
Download the following application note that demonstrates how one of our customers successfully cultivated Streptococcus sp. in Eppendorf a bioreactor system:
- Application Note 337: A BioFlo® 120 bioprocess controller was used to set up a hyaluronic acid production process using Streptococcus
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Clostridium sp.
Clostridium species produce a wide range of products for use in various industries including Botox, ethanol, acetone, and butanol. Due to their production of sustainable biofuels, Clostridium species are of particular interest for the biofuels industry.
Download the following application notes that demonstrate the successful cultivation of Clostridium sp. in Eppendorf bioreactor systems,.
Download the following application notes that demonstrate the successful cultivation of Clostridium sp. in Eppendorf bioreactor systems,.
- Application Note 358: The BioFlo® 120 control station was used for anaerobic fermentation of C. beijerinckii.
- Application Note 434: BioBLU® f Single-Use Bioreactors were used to scale up anaerobic fermentation of C. beijerinckii from 250 mL to 1 L.
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Escherichia coli
E. coli is one of the most widely used organisms for bacterial fermentation in biotech and biopharma.
Download the following application notes that demonstrate the successful cultivation of E. coli in Eppendorf bioreactor systems:
Download the following application notes that demonstrate the successful cultivation of E. coli in Eppendorf bioreactor systems:
- Application Note 340: E. coli fermentation in the BioFlo® 320
- Application Note 307: E. coli fermentation in the BioFlo® 120
- Application Note 462: E. coli fermentation in the SciVario® twin
- Protocol 032: E. coli fermentation in DASbox® Mini Bioreactor System and DASGIP® Parallel Bioreactor System
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Lactobacillus sp.
Lactic acid bacteria (LAB) are known for their production of lactic acid in food and beverages. One of the most widely utilized probiotics, LAB is also regularly used to treat human health issues like diarrhea, vaginal infections, and skin diseases like eczema.
Download the following application notes that demonstrate the successful cultivation of Lactobacillus sp. in Eppendorf bioreactor systems by our customers:
Download the following application notes that demonstrate the successful cultivation of Lactobacillus sp. in Eppendorf bioreactor systems by our customers:
- Application Note 299: A DASGIP® Parallel Bioreactor System was used for optimizing Lactobacillus sp. fermentation
- Application Note 412: BioFlo® 120 and BioFlo® 320 bioprocess controllers were used for microaerobic fermentation of Lactobacillus acidophilus to mimic the natural physiology of the human gut.
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