Two steps forward, one step back – the path to perfect fermentation
Fermentation is part of the basic process of biotechnological production. If fermentation approaches are to be scaled up from laboratory to production scale, some approaches go from the already larger format back to the small laboratory format until the large-scale fermentation approach is ready. We asked two experts from BRAIN about fermentation, planning, the technical challenges and trends.
The coronavirus pandemic has given new life to many hobbies. Among other things, many people have discovered baking with sourdough as a welcome pastime (and definitely also as a challenge). Flour, air and water – not much more is needed! The underlying biochemical process that comes into play when microorganisms from the air come together with flour and water and are given a little time is spontaneous fermentation.
In contrast to such spontaneous fermentation, targeted fermentation processes are planned down to the last detail. Special technical equipment is required and, of course, a great deal of experience. At BRAIN AG in Zwingenberg, fermentation is carried out on a small scale in the laboratory, occasionally also on a somewhat larger pilot scale of up to 200 liters. The British enzyme specialist Biocatalysts handles larger production-scale fermentations for BRAIN Group customers.
We asked bioprocess specialists Dr. Christian Naumer and Sebastian Hohlreiter from BRAIN AG about the process of targeted fermentation and what technical details need to be taken into account.
Sebastian, what happens from a biochemical point of view in a fermenter?
SEBASTIAN: Basically, microorganisms act like us (laughs): They eat to live. In addition to nutrients, most of them, like us, need air - which is also provided in a fermenter or a bioreactor.
Technically, a fermenter is nothing more than a closed vessel in which stirring takes place. We add a specific microorganism (MO) and it uses the supply of organic substances, nutrients and trace elements present in the liquid medium to produce energy. Enzymes inside the cells convert the substrate, producing ATP, the energy of the cells. The organisms use this energy to reproduce and to produce target molecules. The metabolic products either remain in the cell or are released into the medium.
Under certain conditions, which can vary from organism to organism, the microorganisms then produce valuable substances from the substrates. We or our customers are particularly interested in these. These can be enzymes, proteins or small molecules, which are then used in industrial processes and, in the case of enzymes, replace chemical reactions with biochemical reactions. The production of such valuable substances is artificially induced in the fermenter, for example, by introducing genes into the microorganisms. The genes are switched on by induction with a specific substance or by the lack of a nutrient.
How do you plan a fermentation before you get started?
CHRISTIAN: Ideally, the best possible conditions for the organisms are provided in the fermenter: e.g. with regard to the type and concentration of the nutrients offered, the temperature, the pH value or the oxygen level. Basically, therefore, the first thing to do is to obtain as much information as possible regarding the growth behavior and requirements of the strain. In which medium does it grow particularly well? At which pH value? Does it need a special chemical compound to grow?
Then it is investigated whether batch or fed-batch fermentation is more suitable. In the case of fed-batch fermentation, a "feeding strategy" is then defined. Other process conditions must also be defined, i.e. temperature, stirrer speed (and control), gassing rate and duration of the fermentation process. Much of this information is described in scientific publications, but especially for special cases or poorly described organisms, much of it must be determined in preliminary experiments. Thus, it may well take some time before we go into the fermenter with a new strain for the first time.
Apart from volume, what distinguishes a lab-scale fermenter from an industrial-scale fermenter?
CHRISTIAN: Small-scale fermenters offer much better mixing because it is possible to turn the stirrer very quickly, e.g. at 2,000 rotations per minute (rpm). On a large scale, the maximum of about 200 rpm is quickly reached. As a result, not only the mixing but also the introduction of oxygen into the medium decreases significantly. These are the most meaningful differences between the two sizes - and thus also the most important. In addition, the size on an industrial scale means that cooling the bioreactor becomes more demanding and also more expensive.
The large fermenters are usually stainless steel vessels, whereas glassware is often used in the laboratory. This results in different starting conditions for sterilization and cleaning. On the small scale, these are still carried out by hand and the fermenter is sterilized in an autoclave, a type of pressure cooker. The large fermenters, on the other hand, are sterilized with hot steam or chemically. Here, special programs are used for cleaning, which make it possible to clean all pipes leading to and from the fermenter. Otherwise, the laboratory-scale fermenters are equipped with the same sensors as the industrial-scale ones, so that the same data can be collected.
What are the different types of fermentation from a technical point of view?
SEBASTIAN: There are three different types, batch, fed-batch and continuous fermentation. In batch, cultivation continues until the nutrient source is exhausted or used up and there is a deficiency. After that, the organisms can no longer grow and enter the so-called stationary phase until they eventually die. As a rule, fermentation is terminated as soon as it is recognized that the energy source has been exhausted.
If, after the nutrient supply has been exhausted, the deficiency is remedied in the form of an influx (= feed), this is referred to as the fed-batch process. This feed can be added in different ways so that the growth of the cells is linear, exponential or constant. The option of a feed usually generates significantly more growth and product. This is the most commonly used process in our laboratory.
In the continuous process, nutrients are constantly fed, and the amount that enters the reactor is removed through an outlet. This gives a balance between inflow and outflow and the volume in the vessel remains constant. This allows cultivation for a very long time, and influences on cell growth can be mapped or observed.
Which microorganisms does BRAIN use in fermentations, for example?
CHRISTIAN: We work with a wide variety of bacteria, such as Bacillus subtilis/licheniformis, Pseudomonas putida and Escherichia coli. But also yeasts and fungi, such as Picha pastoris, Kluyveromyces lactis and Aspergillus niger. We also cultivate more exotic organisms, including Streptomyces, Aceotbacterium woodii and Rhizomucor mihei.
If a certain approach is fermented to BRAIN's satisfaction on a laboratory scale and this approach is then scaled up at a partner within the BRAIN Group: How does such a scale-up proceed? What difficulties can arise in the process?
SEBASTIAN: Initially, the process has to be developed and established on a laboratory scale, i.e. with one to ten liters. Then the process is transferred to the next size. At our site in Zwingenberg, our 200-liter reactor is then used. After that, the optimized process is well documented and transferred, for example, to our subsidiary Biocatalysts, where cultivation can be carried out on an even larger scale (500 – 8000 liters).
During such a tech transfer, one is in constant exchange with colleagues in the other companies. The task now is to apply the foreign conditions to one's own process in the best possible way. Since not every laboratory is equipped in the same way, problems of the most varied kind can arise here. If, for example, the partner does not use the same filter membrane that we took for cell separation, there may be different results in terms of yield. Then we at BRAIN go back to the laboratory scale and test alternatives, for example cell separation by means of separation or centrifugation – of course with regard to the possibilities available to the manufacturer.
Other problems can arise with the methodology for preparing liquid media, or if the partner manufacturer wants to dispense with a certain medium component, as it is known to have an influence on their own reactors. Thus, during the tech transfer or scale-up, the process is defined more and more specifically to the manufacturer's specifications until the first processes are run there on a large scale. A lot of fine-tuning is also done within the transfer. The most important factor in all of this is communication. The whole thing can only work if people speak correctly and at the same level.
Do you see a trend in fermentation in the future?
SEBASTIAN: From a technical point of view, I see several possibilities. Especially in pharmaceutical biotechnology, single-use bioreactors are very popular. These are, as the name suggests, reactors made of plastic that are disposed of after a single use. This eliminates the need for costly cleaning and sterilization procedures. Process reliability is also increased due to the minimized risk of cross-contamination. Of course, these advantages are offset by the disposable aspect.
CHRISTIAN: As far as products from fermentations are concerned, the production of animal proteins in fermentations, so-called "precise fermentation," is a future technology that will become increasingly important in the coming years.
Thank you both for the interview!
Dr. Christian Naumer, who holds a PhD in biotechnology, began his industrial career at BRAIN in 2005 as a research scientist. Since July 2019, he has headed the Bioprocess Development Technology Unit.
Sebastian Hohlreiter started his professional career at BRAIN in 2018 after completing his bachelor's and master's degree in biotechnology. He handles the development of fermentation processes and the transfer of these to contract manufacturers.
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