What is a bioreactor?

Bioreactor is a vessel in which raw materials under controlled conditions are converted into products by action of living cells (microorganisms, mammalian, plant and stem cells, tissues and algae) or by the cellular components such as enzymes.

The difference between bioreactor and fermenter

Bioreactor and fermenter are similar terms, but with a distinct difference. The term bioreactor often relates to the cultivation of mammalian, plant and stem cells.

If the application is the cultivation of a bacteria, yeast or fungi, then the term fermenter is used. It will not be a mistake to use the term bioreactor in such cases as well, but in the case of cell cultivation only the term bioreactor is used. The name fermenter is associated with reactors in which fermentations are carried out, i.e.  metabolic processes  that produce chemical transformations in organic substrates through the action of enzymes. The main difference between the bioreactors used for cell and microorganism cultivation is in the mixing and aeration requirements, as well as height and diameter ratio H/D. The mixing environment for microorganism cultivation is usually intensive, with effective dispergation of gas bubbles, and the aeration rate is between 0.5- 3 vvm (volume cultivation media/volume air flow per minute). However, cell cultures require gentle mixing and aeration rate is beween 0.01-0.1 vvm. In turn, the optimal H/D ratio of the vessel for microorganism cultivation is 3:1, but in the case of cell cultures it is 2:1.

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The classification of bioreactors

Generally, bioreactors can be characterized in two ways:

  1. The cultivation principle;

  2. The operation mode.

Cultivation principles of microorganisms are subdivided further: submerged, immobilisation and solid state.

The cultivation principles

Submerged cultivation

Inoculation of the microbial culture into the liquid medium for generation of the desired product is known as submerged cultivation or fermentation. Aerobic and anaerobic fermentation processes are the two separate fermentation processes.

In submerged fermentation (or cultivation) the cells of the producer (microorganism) are supplied with nutrient medium and oxygen (in the case of aerobic process) in the entire working volume of bioreactor by mixing and aeration. This makes the process highly economical. The bioreactor creates favorable conditions to accumulate a large amount of actively functioning producer biomass and in turn the target product.

As an example, relating to the history of biotechnology, it can be pointed out that replacing surface fermentation (in flasks and bottles) with submerged fermentation made it possible to increase the production of penicillin in a short time, especially urgently needed during the Second World War.

The submerged fermentation can be aerobic or anaerobic. For example, antibiotics and enzymes are produced through aerobic fermentation, which involves the incorporation of oxygen into the liquid medium, while butanol production proceeds under conditions of anaerobic fermentation, wherein the oxygen influence will have a inhibitory effect. Certain fermentation processes, such as ethanol production, use facultative anaerobic organisms like Saccharomyces cerevisiae, which may grow in the presence of oxygen and produce cell biomass before switching to anaerobic mode during the ethanol fermentation phase. Enzymes (amylases and proteases, amylases, and so on) are often made through aerobic submerged fermentation.

Submerged fermentation processes can be differentiated depending on the target product. The target product can be biomass, ferments or low-molecular compounds (for example, ethanol, methanol, acetates, oxalic and formic acids). Metabolites can be primary or secondary. A primary metabolite is a type of metabolite that plays a direct role in normal development, growth, and reproduction. Some common examples of primary metabolites are lactic acid, and certain amino acids. Secondary metabolites are generated toward or at the conclusion of the stationary phase of growth and do not play a role in growth, development, or reproduction. Atropine and antibiotics like erythromycin and bacitracin are examples of secondary metabolites.

  

Fermentation processes can finally be differentiated in terms of technology and the type of target product. The target product can be biomass, an individual high-molecular substance (for example, as an enzyme - constitutive or inducible) or low molecular weight metabolite. Metabolite, in turn, can be primary or secondary. Thus, the need for inductors and precursors, as well as the time of their introduction into the medium, depends on the target product. The biosynthesis of secondary metabolites is characteristic of certain stages in the development of the producer's culture and is stimulated in stressful situations. In this regard, the introduction of inducers and precursors is mandatory in the case when the target of the fermentation process is a secondary metabolite.

The biomass accumulation curve usually correlates with the accumulation curve of primary metabolites and does not coincide with the accumulation curve of secondary metabolites.

Submerged fermentation is best suited for microorganisms such as bacteria and yeasts that require high moisture.  Another advantage of this method is that product purification is simplified.  Submerged fermentation is most commonly employed to extract secondary metabolites that must be used in liquid form.

To carry out submerged fermentations are used as most typical stirred tank, bubble column, airlift type bioreactors, as well photobioreactors and membrane bioreactors in special applications.   

The operation modes

Batch

Batch culture is a closed system in which the growth rate of biomass tends to zero due to depletion of the substrate and accumulation of inhibitors. Such systems are always in an unstable state.

There six growth phases of microorganisms during batch cultivation process.

1. The lag phase or induction period begins after inoculation into the nutrient medium of the microorganism and is the period of their adaptation.

During this phase, there is a reorganization of the micromolecular and macromolecular components of the microbial culture, the synthesis or suppression of enzymes or structural components of the cell. This phase, depending on external conditions, can be in ranges from 1 hour till 3 or more hours. During this phase, the cell mass can change without changing the number of cells.

2. The lag phase enters the exponential growth phase. This is a period of rapid accumulation of biomass and reaction products.

3. The phase of linear growth is characterized by balanced growth in the steady state, i. e. the growth rate remains constant throughout the entire cultivation process, and the chemical composition of the culture liquid changes, since nutrients are consumed and metabolic products are produced. As a consequence, the environment surrounding microorganisms is constantly changing, but the growth rate in a wide range of nutrient concentrations does not depend on them.

4. The phase of linear growth is replaced by a period during which the growth rate of the culture decreases to zero - this is the phase of growth retardation.

5. Further, the growth of the culture can pass into a fairly stable stationary phase, while the rate of death of microorganisms is compensated by the rate of increase in biomass.

6. With the complete depletion of the nutrient medium (substrate) and a significant accumulation of growth-inhibiting products, significant physiological changes in the culture (lysis) occur and the so-called phase of culture withering begins.

bioreactor cultivation process types

Solid state fermentation 

 

Solid state fermentation (SSF) is carried out using solid phase substrate. The microorganisms are growing on a solid substrate in absence or near absence of free water.

 

The substrate must generate enough moisture to support growth and metabolism of the microorganism. SSF are applied for the production of fermented food products, for example, bread, meat cheese, pickles and yogurt. Using SSF can be recycled agro-industrial residues to obtain, for example  enzymes, organic acids, food aroma compounds, biopesticides, mushrooms, pigments, xanthan gum and vegetable hormones. SSF requires less instrumentation and design of bioreactors are relative simple. However, scale-up is bothered, because it is difficult to ensure precise monitoring and control, and can not be controlled environmental conditions of the microorganisms. SSF are long, because the growth rate of microorganisms on solid substrate is slow. There are the processes, which successfully can realized only by SSF. For example, the sporulation of some fungi can attained only by SSF since these fungi do not sporulate in liquid media.    

 

For SSF are used horizontal drum, tray-type, packed-bed and bench scale bioreactors.

Immobilization

Immobilization means the binding of an enzyme to an insoluble carrier while maintaining the functionality, i.e. the catalytic activity of the enzyme.  The need of immobilization is determined by the fact that in many applications the end product must be completely free from enzyme resudues in order to avoid immune reactions.

 

The immobilization of enzymes not only significantly increases their stability, but allows the long-term use of one batch or series of industrial biocatalysts. The concept of "immobilization of a biological object" means the physical separation of a biocatalyst and a solvent, in which molecules of the substrate and reaction products can freely penetrate from a liquid to a solid medium, and vice versa. In other words, the substrate in the flow of the solvent is supplied to the bio-object associated with an insoluble carrier, and the reaction product in the flow of the solvent is removed from the bio-object and is used as the target product.

Enzymes, as well as entire cells, can be immobilized. The immobilization, i.e., the fixation of cells onto a carrier, for example, in ethanol produc­tion has several advantages. The cells could be reused and have extended lifespan. Some of the traditional purification procedures are not required because the desired final product is essentially free of biological substances and organisms. Inclusion of cells in gels, in which a cell solution is combined with gel-forming chemicals, is one of the most frequent immobilization procedures. Small molecules such as glucose can pass through the gel pores to reach the cells while their metabolic products (alcohol and carbon dioxide) can exit the beads. The living yeast cells so remain intact.

Various immobilization techniques such as the attaching of cells in stable porous gels (e.g., alginate, collagen, chitosan, agarose, cellulose, κ-carrageenan, or gel-matrix polymers such as polyacrylamide-hydrazide) or hydrogels or immobilization in solid macroporous carriers has been established and is used on both laboratory and industrial sizes for a variety of applications, including the food, dairy, and beverage sector, medication production, wastewater treatment, agriculture, and biodiesel generation. In the case of the production of pharmaceutical preparations, the target substance will not contain components of the culture liquid (mycelium, products of partial lysis of cells, components of a complex nutrient medium, etc.), which greatly facilitates the task of isolating and purifying the target product, guarantees the absence of proteins and other harmful impurities.

The economic advantages of using immobilized biological objects in production conditions are undeniable. The use of immobilized systems makes it possible to make the conditions of biosynthesis more standard, and the entire production more compact. The resulting biological object works for a long time. At the same time, less raw materials are consumed per unit of production.

The application problem can be that the cells may contain numerous catalytically active enzymes, which can cause unwanted side reactions and the cell membrane itself may serve as a diffusion barrier, thus reducing productivity. It is difficult in immobilized cell bioreactors to control the physiological state of microorganisms, and due to the process variability and flexibility can not be ensured.

Immobilized cell bioreactors divide into stirred tank, fixed bed, fluidized bed, moving bed, packed bed and membrane reactors.

Fed-batch

Fed-batch is based on feeding of a growth-limiting nutrient substrate to a culture. Cell growth and fermentation process can be controlled by varying the feeding strategy.

Usually fed-batch is started with batch fermentation phase until consumption of one or more substrates and/or inducers into a bioreactor.  The fresh medium can be added by using different feeding regimes. Feeding can be added via a fixed volume or variable volume of a fresh medium or substrate only during the time course of the process. This feeding can be continuous or exponentially or pulses over a short or long period during the fed-batch phase.

When the target product is positively tied with microbial growth, fed-batch fermentation is highly advantageous for bioprocesses aiming for high biomass density or high product yield. A typical fed-batch process for the production of a product takes place in 3 stages. First, in the batch mode, the biomass is growth up to such a concentration that makes it possible to continue the process with a limit on the substrate (without the accumulation of the substrate in the medium). The second stage is the stage of biomass cultivation, when the media is fed with a substrate that promotes the rapid growth of the culture (glucose, sucrose or glycerin). The last stage is the stage of product synthesis (recombinant protein or other substances) when a biosynthesis inducer is introduced into the medium. This may be a substance that activates gene transcription (for example, IPTG) or a substrate that is an auto-inducer (for example, methanol for P. pastoris or lactose for E coli).

 

The simplest way of feeding strategy is calculation of time dependent adjusted feeding profile. This feeding profile usually is calculated using mathematical models. The feeding regime must be controlled by operator to prevent the repressive effects of high substrate concentrations and avoids catabolism repression. The control of fed-batch is problematic, because there are not available reliable biomass and substrate concentration on-line measurement methods.

By using adjusted feeding profiles in reality operator have to always these profiles during a fed-batch to correct. This is due to impossibility of providing fully repeatable fermentations. At certain stages of the fermentation process, it is possible to successfully perform the automatic feeding based on the dissolved oxygen DO sensor readings. But there are several problems here. Firstly, in this event it is impossible to control the DO concentration by rotation speed and/or oxygen-enriched air supplied to the aeration, and secondly, when relatively high biomass densities are reached, it is impossible to implement this control in an optimal version.

 

Aforementioned problem could be solved by model based fed-batch control. The ready solutions are yet not available in the market. Usually model based control systems are developed for specific applications. There are some attempts to develop systems available for wider applications (for example, https://www.bioreactors.net/model-based-fed-batch-control).

Generally model based control functions in the following way: During the fermentation process, samples are taken in order to input the current results of the tests on biomass and substrates in the software. Performed the input of the results of the tests, the software performs automatic comparison of these results with the results of calculations according to the adopted mathematical model. If the deviations are above the set standards, the software performs calculation of the new feeding profile. The updated feeding profile is automatically uploaded into the process controller PLC, and the substrate feeding is further implemented according to the new profile (up to a following update). Mathematic model is implemented as PC program, and this program is connected to PLC with the help of OPC server.

Continuous

Continuous cultivation is characterized by the constant addition of fresh nutrient medium to the bioreactor and the constant selection of either a suspension or a spent medium. Continuous culture is an open system that seeks to establish a dynamic balance. In this way, constant environmental conditions in the cultivation media can be ensured. Continuous cultivation is applicable if the produced product has the appropriate demand potential. The other typical application of continuous cultivation can be in wastewater treatments using wastewater as in-flow substrate. 

Continuous cultivation is applied if is necessary regulary amount of product. structurally more complicated and requires additional automatic control, since it is associated with the introduction of additional devices into the bioreactor connection schema.

In a batch culture, conditions change all the time: the density of the culture increases, and the concentration of the substrate decreases. However, it is very often required that cells can be in the phase of exponential growth for a long time at a constant concentration of the substrate under unchanged other conditions. This can be achieved if a new nutrient solution is continuously introduced into a vessel containing a cell culture and at the same time an appropriate amount of cell suspension is removed from it.

In the practice of microbiological research, two types of open flow cultivation are widely used: chemostat and auxostats methods.

The chemostat method of cell cultivation is based on the use of a bioreactor, into which a nutrient medium is supplied at a constant rate and at the same time (for example, drainage according to the level) the cell suspension is taken. At the same time, the volume of the grown suspension remains constant. The growth of the culture in the chemostat is controlled by the concentration of the substrates. The stability of the system is based on this limitation of the growth rate by the concentration of one of the necessary substrates.

The auxostats are close-loop systems, which are controlled by feed-back regulation of some state variable, e.g. biomass or a substrate concentration or pH. Depending on the principle of operational control the auxostats can be classified as a turbidostat, a nutristat, or a pH-auxostat.

In a turbidostat the feed rate is adjusted by an optical density (turbidity) controller so that a constant biomass concentration is maintained over time. Under conditions of nutrient excess, the turbidostat provides the process close to maximal growth rate. The application problem of turbidostat is connected with some technical difficulties of sensor readings, e.g. fouling of the sensor due to microbial growth on its surface, disturbances in signal transfer by air bubbles or coloured and particulate media.

Nutristat operation is based on the measurement and control of substrate concentration by feeding of substrate. The use of nutristat is restricted due to the lack of suitable analytical tools for on-line measurement of most relevant substrate concentrations.

The pH-auxostat is based on measurement of the pH which is often correlated to the biomass production rate, but easier to measure and control as turbidity and substrate concentrations. The signal from the pH sensor is used to control the medium in-flow in a titration mode so that addition of the fresh medium brings the pH back to the setpoint, and the same amount media is taken away from bioreactor in out-flow. The pH-auxostat method is applicable for the microorganisms with growth that causes changes of the medium pH.

The application example of bioreactors

The cultivation for acquiring vaccines against the SARS-CoV-2

Recently, considerable effort has been put into the development of a vaccine against the SARS‑CoV‑2 The production process of all vaccines involves the use of bioreactors. For example, the approach for creating two types of coronavirus vaccines, which are already clinically confirmed, are the following:

RNA vaccines are made using a new technology that was previously used only in veterinary medicine. No RNA vaccine has yet been approved for use in humans. This vaccine contains a viral molecule similar in structure to the vaccine contains a viral messenger RNA (mRNA) molecule, which is similar in structure to human mRNA. Upon entering the human cells, the mRNA is used by the ribosomes in producing a viral protein. The mentioned protein stimulates an immune response in the human body, thus generating natural resistance to the virus. This method is used in the case of Pfizer-BioNTech and Moderna vaccine operation.

To manufacture this type of vaccines a host organism is required, which can produce large quantities of viral mRNA through cultivation. The most widely applied organism for such applications is the Escherichia coli bacterium. The process can be performed in stainless steel bioreactors, thus it is possible to cultivate the bacterium in up to 10 m3 (and even larger) working volume bioreactors. This means that the process is relatively easy scalable. Although, this type of cultivation process cannot be carried out in disposable (single-use) bioreactors, as they usually cannot provide sufficiently intensive mixing and aeration, which is necessary for E.coli growth and mRNA production. The other type of vaccines use a weakened type of adenovirus, which carries a specific viral protein for triggering an immune response.

These vaccines are examples of non-replicating viral vectors, using an adenovirus shell containing DNA that encodes a SARS‑CoV‑2 protein. The viral vector-based vaccines against the coronavirus are non-replicating, meaning that they are incapable of producing new viral cells, but rather produce only the antigen which elicits a systemic immune response. Vaccine of this type are the Oxford–AstraZeneca COVID-19 vaccine, Sputnik V (Russia),  Convidicea (China) and  Johnson & Johnson's Ad26.COV2.S.

For the manufacturing of these types of vaccines the cultivation of mammalian cells is used. In a such cultivations single-use bioreactors can be applied, allowing relatively easy production plant expansion in terms of simultaneously operating bioreactors. The maximum working volume of a stable single‑use bioreactors on the market today is about 2000 liters. Although, relatively recently the announcements of larger volume single-use bioreactors have been observed. For example, ABEC, a global provider of integrated solutions and services for biopharmaceutical manufacturing, recently announced the availability of single-use bioreactors with working volumes of up to 6000 liters.