When we think about fermentation design we are considering what the properties are that makes for the ideal fermenter. Fermentation is primarily about supporting growth of a microorganism usually as an optimum or a maximum level of growth. To achieve this the fermenter must operate aseptically so that no other microbes contaminate the biomass, It must have an appropriate level of aeration and mixing or agitation. We want it to be energy efficient and consume as little power as possible. It needs to be controllable in terms of pH and temperature and there must be sampling facilities.
The other key features are that operation is simple and straightforward and that services are available such as steam etc.
Fermentation in a bioreactor has many different forms. The most common that you will come across is a simple batch fermentation of which there are many variants, the fed-batch fermentation, the continuous fermentation and the perfusion bioreactor. These four types vary in terms of how substrate is replenished or not and how it affects the growth of the biomass and the production of desired products. These are also liquid fermentations.
The solid-state fermentation is one which requires a solid-phase for fermentation. It includes composting and the production of vinegar as examples but is discussed elsewhere.
One of the best textbooks on the subject of fermenter design and type is ‘Biochemical Engineering Fundamentals‘ by Bailey & Ollis (McGraw-Hill Int. Edt. [New York] 1986)
Fermenter Design
A typical fermenter will be a vessel made of glass and preferably borosilicate, steel (316 L) and even plastic. Some have flat or rounded bases but designed in such a way that there are no dead volumes or points where the addition of sensors creates a different environment to the bulk environment.
316 L stainless steel is highly corrosion resistant as well as resistant to chlorine. It has a composition of 65% steel, 16-18 % chromium which provides corrosion resistance, 12-14 % nickel for improving ductility and formability, a small amount of molybdenum (2-3%) and finally carbon (0.03 % max.) which helps in reducing sensitization during welding.
For smaller scale fermenters, borosilicate glass is inert, easy to clean, robust and easy to see through. It is mainly SiO2 (81%), B2O3 (13%), Na2O and K2O (4%) and 2% Al2O3. Glass thickness can be variable and depends on bubbles in the glass material.
The internal surfaces must be as smooth as possible. Likewise, on scale-up the geometries should be similar between small and large vessels.
Most fermenters are designed with a top plate and a number of ports which are openings. These are for a range of standard fittings such as sensors, nutrient feed and exit pipes, a stirrer (impellar), samplers and harvesting pipes. metal with met
The seals on the fermenter and attendant parts need to be aseptic. These seals can be between glass and glass, metal and glass, metal with metal etc. Any junctions are best served by a compressible gasket such as a lip seal or an O ring. These seals are contained in groove systems for optimal adhesion. The seals are designed to accommodate expansion and contraction of the various vessel materials.
Aeration is needed to provide microorganisms in a submerged culture with sufficient oxygen for their metabolic requirements. Agitation in the form of mixing is needed to generate a uniform suspension of the microbial cells and ensure fresh nutrient is supplied at all times. Mechanical agitation is always needed for fungal and actinomycete fermentations.
The structural components needed for aeration and agitation are:
- impellar/agitator
- stirrer glands and bearings
- baffles
- aeration system which is a sparger.
The Impellar
Impellar design can be complex but its main purpose is to mix the bulk fluid and any gas-phase so that cells, nutrient and gas are dispersed uniformly. It should also disperse any heat and move solid particles throughout the environment of the vessel.
Baffles
Baffles are used to help with agitation and reduce foaming. Most often four baffles are incorporated into the vessel to prevent vortexing and improve aeration efficiency. These are often metal strips that are roughly one-tenth the diameter of the vessel and attached radially to the vessel wall. The baffles are of such a size to minimise microbial growth on their surface and must be so designed so there are no deqad spots for aeration or mixing.
Gases Used
Gas is sparged into the bulk liquid of a fermenter. This can be air, oxygen, nitrogen and mixtures. Vents are needed to avoid the build up of pressure in the vessel and for easy removal of gases especially exhaust. In many cases, carbon dioxide gas is vented off when it is a byproduct of the reaction.
The aeration sparger can be of three types: a porous sparger which generates tiny bubbles for optimal gas transfer, an orifice sparger which is a perforated pipe or a nozzle sparger which is an open or closed pipe. Sometimes the sparger is combined with an agitator because gas bubbles are very effective at mixing liquids too..
Sensors
- pH
- oxygen (pO2 ), air, carbon dioxide and nitrogen pressure
- temperature
- biomass.
pH Control
A set pH is most often required. Control is achieved through automatic addition of either 2N NaOH or HCl.
Temperature control
Temperature control needs to be regulated firmly. Most fermenters are jacketed to allow for temperature control of the fermentation liquid. Some fermenters are placed in thermostatically controlled baths or use internal heating coils. In some cases the jacket is silicone through which water is circulated. The silicone jacket can take the form of rubber mats wrapped around the vessel and heated electrically.
Water is supplied at various temperatures with steam/water mixes used for higher temperatures. The most common temperature range is between 15 and 40°C.
The Feed Ports
The feed ports are usually sited on the top plate. These are for addition of nutrients, inoculum and other supplements, Sampling ports are needed for sample removal. There should also be ports for acid and alkali additions as in pH control. These are usually supplied via peristaltic pumps after aseptic connection using silicone tubing. In larger fermenters, nutrient reservoirs and associated piping are integral parts of the fermenter and can be sterilized with the vessel. The silicone tubes can be autoclaved separately.
Fermenter Sterilization
The fermenter must be designed for steam sterilization. Some may be small enough to be autoclaved. It’s likely any steam sterilization will be under pressure. The medium for fermentation should be sterilized in the vessel or treated separately and then added aseptically. The steam should be introduced separately. It also needs to also be able to vent from within the internal volume of the fermenter.
The air supply should be sterile. Sterile air in large volumes is always needed for aerobic processes. Filtration is simpler and less costly than heating and cooling of air.
The sterilization of the exhaust gas from the fermenter is best achieved using a 0.2 micro filter on the outlet pipe. Beware though that aerosol formation can occur in the fermenter with solids and moisture plugging the filter. Always check the filters contain only non-viable cells.
Foam Control
When a fermentation broth with cells is sparged and aerated it has a tendency to produce foam which clogs supply and exit holes, blocks filters and tubing. To avoid this, foaming is minimised by adding an antifoam or designing baffles which break up the foam.
The other rarer situation is siphoning caused by differential pressures and causes loss of contents .
The Batch Fermentation
The most commonly operated fermentation is the batch fermentation. It is operated discontinuously meaning that once the fermentation has started, there is no replenishment of substrates and the biomass grows to a point where it is harvested completely or the fermentation is stopped at the optimum point of production of a secondary metabolite.
The fermenter/bioreactor is completely cleaned between each batch operation. A new batch production is then started.
There are 5 key steps in the process. Firstly, a fresh batch of medium is prepared and added to the fermenter. The fermenter and the medium are sterilised fully including the monitoring equipment. Once it has all cooled down, an inoculum is added. The fermentation progresses to a point where the biomass is harvested or the fermentation medium containing substances of interest is separated and processed further. The whole procedure is repeated.
The key features of a batch fermentation system is that it is by far and a way the simplest type of fermentation operation. The contents as well as the fermenter is sterilised fully so all the nutrient for fermentation must be in place before that sterilization step.
The maximum levels of carbon and nitrogen are limited by the inhibition of cell growth. Likewise, biomass production is limited by the carbon to nitrogen (C/N) ratio and load and the accumulation of toxic waste products which eventually put a halt to further biomass growth.
The main benefits of the batch fermentation are that the end product which could be the biomass or a substance produced by that biomass is obtained in significant quantity over a fixed period of time. A good example is a beer or wine fermentation. The end product too may have a limited shelf-life and so be collected soon after it has been fermented. The other feature is that batch fermentations work well when the product is produced only in the stationary phase and not in any of the growing phases.
Manufacturers of bench-top batch fermenters include Infors’ Minifors 2 which comes in 1.5L, 3 L and 6 L vessel sizes.
The Continuous Fermentation
In a continuous process, the nutrient is continuously added to the fermenter at a fixed rate. The microbes are continuously maintained in a logarithmic phase and not a stationary phase. Any products are recovered from the fermentation medium continuously.
Sometimes this fermentation is known as a ‘flow through’ fermentation.
Whilst the continuous fermentation has many benefits, sterilization of the media is more complex and often surprisingly difficult to achieve. It is unfortunately more prone to contamination too as well as suffering with cell degeneration as well as cell wash-out.
The Fed-Batch Fermentation
The 3rd basic type is the fed-batch fermentation which is viewed many ways depending on how it is operated. It is considered an intermediate type between batch and continuous fermentation. For some experts it is actually an extended type of batch culture where the medium containing the substrate is fed into the fermenter without harvesting any product. The nutrients are sterilised and added in incremental amounts. At the end of the fermentation, the biomass and product is harvested as in a batch culture.
We can also view it as a semi-continuous operation or as a semi-closed system of cultivation. The main consideration though is that a batch fermentation is a steady-state process whereas a fed-batch process is an unsteady-state process. This is because the liquid volume in the fermenter is allowed to increase with time whilst the withdrawal of products is not continuous.
Initially, the fermentation operates in a batch manufacture. The initial medium concentration is relatively low. When the nutrient is used up (consumed) which is often the carbon source, more substrate is fed into the reactor following a predetermined protocol. The medium constituents are added in increments in a controlled manner which often results in high biomass and better product yields. However, the fermentation is still limited by the accumulation of toxic end products.
The products – biomass and secondary metabolite are harvested as in a batch fermentation after the fermentation has completed. It is very popular in industry for the production of bakers yeast and antibiotics. Good examples of fed-batch fermentations and the way they are operated are demonstrated in the production of the amino-acid l-tryptophan from the substrate glycerol (Trondle et al., 2018), production of 3-dehydroshikimic acid (Li et al., 1999) or production of pyruvate (Zelić et al., 2003). These are all fermentations using recombinant forms of the bacteria Escherichia coli.
The Perfusion Bioreactor
The Perfusion bioreactor is a very specialised fermenter for a number of applications where gentle fermentation conditions are needed. It is a more advanced form of fed-batch fermentation and is highly regarded for the benefits it brings especially in the cultivation of mammalian cells and for producing high titers of expensive proteins such as monoclonal antibodies (Chu & Robinson, 2001). Whilst batch and fed-batch fermentation is still the most widely used production method for mammalian cells it is perfusion based fermentation which is increasing in commercial uptake.
The perfusion method relies on providing a constant environment which favours cell growth with continuous by-product removal and nutrient addition. A comparison with both batch and fed-batch approaches means the perfusion method prolongs a healthy culture, a potentially higher cell density and reasonably short residence time for the product in the fermenter. It is the method of choice for producing the blood clotting factor VIII which is generally an unstable glycoprotein (Chotteau et al., 2001).
Sartorius (Germany) are producers of the Flexsafe® perfusion bioreactor bags which have a maximum working volume of 1 to 25 L. These are used in devices such as a 2-dimensional rocking motion bioreactor. This type of bioreactor called BIOSTAT RM 20/50 is also produced by Sartorius.
Aerobic versus Anaerobic Fermentations
The final distinction is the comparison between the aerobic and anaerobic fermentation. An aerobic fermentation relies on oxygen and air to provide a key substrate. An anaerobic type relies on an absence of oxygen for the biomass to grow.
The aerobic fermenter provides oxygen to the microorganisms to support aerobic respiration. It typically includes agitation systems, spargers, or diffusers to ensure proper oxygen transfer throughout the fermentation process. The alternative and commonly used is the anaerobic fermenter because of its industrial significance. In contrast to aerobic fermenters, anaerobic fermenters create an oxygen-free environment, suitable for microorganisms that thrive in the absence of oxygen. These are commonly used in the production of certain products like ethanol and other anaerobic fermentation processes.
Variants Based on Mixing
The Stirred Tank Fermenter is the most common type of fermenter. It consists of a cylindrical vessel with an agitator or stirrer to mix the contents and ensure even distribution of nutrients and oxygen to the microorganisms.
The Tower Fermenter also known as a packed-bed fermenter, is a vertical vessel filled with a solid support matrix (e.g., ceramic rings or sponge-like materials) where microorganisms grow and adhere. The nutrient medium is trickled or sprayed over the support material, providing nutrients to the microorganisms. This type of system has been employed for the production of industrially important products such as ethanol. It is a system that can be operated in both batch and continuous modes. The continuous mode has the advantage of accumulating and retaining high densities of cells as seen in ethanol production (Prince & Barford, 1982).
Trickle-Bed Reactor
The trickle-bed reactor is a three-phase system that contains a packed bed of catalysts with flowing gas and liquid phases. The substrates are supplied in the liquid and gas feeds. Any biochemical reaction occurs on contact of the liquid with the catalyst surface. The packing is critical in this operation.
Airlift Fermenter.
The air-lift fermenter is suitable for particular cells where a gentle mixing action is needed. There is no stirrer in the form of a rod with blades! The shear forces are also reduced compared to impellar-mixed batch fermentations.
The airlift fermenter utilizes the principle of gas lifting to circulate the fermentation broth. Compressed air is introduced at the bottom, creating a two-phase flow that helps in mixing and circulation of nutrients and microorganisms.
The absence of any seals as well as the mixer itself means the potential for contamination is lowered. Most fermenters of this design are usually manufactured specifically by laboratories. There are commercially available large-scale fermenters from 1000 to 2000 dm3. The company Celltech Ltd (UK) operated these fermenters for producing monoclonal antibodies from hybridoma cells.
Fermenter Types: The Modular Bioreactor
Modular bioreactors or fermenters allow the user to scale up from 1 to many types. Up to 36 bioreactors can be operated online with the same or different vessel sizes and typology and also under the same level of control.
Each fermenter can be under the control of a system based on PLC hardware, a ModBus and Ethernet communication which allows for both single and parallel bioreactor control.
In a number of circumstances, bioreactors are operated either singly or in parallel. A number of suppliers such as Sartorius, Infors etc. offer different systems to suit the requirements of the fermentation.
Solida Biotech GmbH (München, Germany) are well known suppliers of these types of equipment .
Fermenter Types : Permanent and Single-Use Fermenters
Generally, most fermentation equipment is designed for sustained use where the vessel is cleaned and sterilized for use on a number of occasions. In recent years, single-use bioreactors have been developed which have the distinct advantages of being presterilised without the worry of cross-contamination from previous fermentations. They have become increasingly popular in fermentation manufacturing,
One of the leading exponents is Thermo Fisher Scientific.
DSM Biologics (Groningen, NL.) have developed XD® Technology which is a process intensification method for producing cell cultures that have a higher cell density compared to a standard fed-batch process. It operates in a continuous media feeding mode with a filtration system to retain cells and recombinant protein within the bioreactor. The reactor is claimed to have a high volumetric productivity. At the end of fermentation, the cell culture is harvested after roughly 14 days as a single batch.
Cell densities of 240 million cells/mL are noted with Chinese Hamster Ovary cells which translates into yields of 11.5 g/L of recombinant proteins when compared to just 1.2 g/L for a fed-batch system. They also produced 13.4 g/L of monoclonal antibodies compared with 1.4 g/L using a simple fed-batch system
Photo-Bioreactor
A photo-bioreactor is designed for cultivating photosynthetic microorganisms, such as algae or cyanobacteria. It incorporates transparent walls to allow light penetration and optimize the growth of photosynthetic organisms.
These are some of the main types of fermenters used in various industries for the production of a wide range of products, including pharmaceuticals, enzymes, biofuels, food additives, and more. Each type of fermenter offers unique advantages and is chosen based on the specific requirements of the fermentation process and the type of microorganisms involved.
References
Chotteau V, Bj€orling T, Boork S, Brink-Nilsson H, Chatzissavi-dou N, Fenge C, Lindner-Olsson E, Olofsson M, Rosenquist J,Sandberg H, Smeds A-L, Drapeau D. (2001) Development of a largescale process for the production of recombinant truncated factor VIII in CHO cells under cell growth arrest conditions. In: Lindner-Olsson E, Chatzissavidou N, Lullau E, editors. Animal Cell Technology: From Target to Market. Proceedings of the 17thESACT Meeting, Tylosand, Sweden, June 10–14, 2001. Dordrecht: Kluver Academic Publisher; pp. 287–292
Chu, L., Robinson, D.K. (2001) Industrial choices for protein productionby large-scale cell culture. Curr Opin Biotechnol. 12 pp.180–187
Li, K., Mikola, M. R., Draths, K. M., Worden, R. M., & Frost, J. W. (1999). Fed‐batch fermentor synthesis of 3‐dehydroshikimic acid using recombinant Escherichia coli. Biotechnology and Bioengineering, 64(1), pp. 61-73 (Article).
Prince, I.G., Barford, J.P. (1982) Continuous tower fermentation for power ethanol production. Biotechnol. Lett. 4, pp. 263–268 (Article).Prince, I.G., Barford, J.P. (1982) Tower fermentation of sugar cane juice. Biotechnol Lett 4, pp. 469–474 (Article).
Tröndle, J., Trachtmann, N., Sprenger, G. A., & Weuster‐Botz, D. (2018). Fed‐batch production of l‐tryptophan from glycerol using recombinant Escherichia coli. Biotechnology and Bioengineering, 115(12), pp. 2881-2892 (Article)
Zelić, B., Gerharz, T., Bott, M., Vasić‐Rački, Đ., Wandrey, C., & Takors, R. (2003). Fed‐batch process for pyruvate production by recombinant Escherichia coli YYC202 strain. Engineering in Life Sciences, 3(7), pp. 299-305. (Article)
Original post 18-08-2022. Post updated to include further examples of fermentation, of fed-batch fermentation using E.coli and of other systems.
Leave a Reply