Bioreactor

Many red biotechnology products are produced in bioreactors, such as this facility for the production of vaccines

Moss bioreactor with Physcomitrella patens

A bioreactor , often referred to as a fermenter , is a container in which certain microorganisms , cells or small plants are cultivated (also: fermented ) under the best possible conditions . The operation of a bioreactor is thus an application of biotechnology that uses or makes usable biological processes ( bioconversion , biocatalysis ) in technical facilities.

Important factors that can be controlled or monitored in most bioreactors are the composition of the nutrient medium (also nutrient solution or substrate ), the oxygen supply , temperature, pH value , sterility and others. The purpose of cultivation in a bioreactor can be to obtain the cells or components of the cells or to obtain metabolic products . These can e.g. B. be used as an active ingredient in the pharmaceutical or as a basic chemical in the chemical industry . The degradation of chemical compounds can also take place in bioreactors, such as B. in wastewater treatment in sewage treatment plants . The production of beer, wine and other products that have been produced for thousands of years also takes place in bioreactors. In contrast to modern applications, these classic examples are usually not referred to as bioreactors, but instead use the historical terms (e.g. brewing kettle in beer production).

A wide variety of organisms are cultivated in bioreactors for various purposes. Therefore, several reactor variants are available in different designs. Typical are stirred tank reactors made of metal, which can have a volume of a few to thousands of liters and are filled with nutrient solution. But also very different variants, such as B. Fixed bed reactors , photobioreactors are used.

history

Fermentation tanks for beer production are also bioreactors

Since brewing kettles in breweries are technically part of the bioreactors, the appearance of the first bioreactors can be equated with the appearance of the first breweries around 5500 years ago. The devices for producing various milk products with the aid of bacteria or enzymes, which have been in use for thousands of years, can also be referred to as bioreactors.

With the further development of biotechnology , especially through significant advances in microbiology in the 19th century and in genetics , molecular biology and genetic engineering from the middle of the 20th century, more and more applications such as in the chemical industry and in the field of pharmaceuticals could be developed. Bioreactors are used in many biotechnological processes .

Operating parameters

The main purpose of a bioreactor is to deliver the highest possible product yields. This is achieved in particular by creating optimal conditions for the organism used in each case. This is adapted to various parameters that prevail in its natural habitat. The type and concentration of the nutrients , the temperature, the oxygen content, the pH value, etc. are important. In addition, a stirrer or other device is usually necessary to ensure that these parameters are set homogeneously over the entire reactor space. In addition to the demands of the organisms, other technical, organizational and economic factors that influence the choice of operating parameters must also be taken into account. Examples are the avoidance of foam formation and the choice of either a continuous or a batch mode of operation .

With the help of probes or sensors , many of these parameters are measured directly in the nutrient medium or in the exhaust air. The course of the process can usually also be assessed using these parameters. The cell density can be determined by measuring the extinction (optical density) , which in turn indicates the amount of product. An alternative is often to measure the concentration of a characteristic chemical compound, e.g. B. the increase in the concentration of a metabolic product or the decrease in the substrate concentration.

At the beginning of a fermentation, the nutrient medium is mixed with a small amount of the microorganism obtained from pre-cultivation . This amount is called inoculum , and the process is often referred to as inoculation . The suspension ( broth ) obtained from the fermentation process is processed in so-called downstream processing in several process steps.

Nutrient supply

With the nutrient medium organisms have all necessary for the growth nutrients are made available. These include the main nutritional elements (macronutrient elements) required in larger quantities , such as B. carbon , nitrogen and phosphorus . Various trace elements (micronutrients) are also required. Depending on the organism, other compounds are necessary that cannot be synthesized by yourself ( vitamins , essential amino acids, etc.). An energy-supplying connection, such as B. often the sugar glucose is necessary (except for phototrophic organisms).

temperature

Organisms have an optimum temperature at which they reproduce best. Exceeding this temperature can lead to irreversible damage due to denaturation of the proteins, and falling below this leads to lower metabolic speeds and thus to longer process times. The temperature control is implemented by heating and cooling circuits. When the reactor is started up, the entire contents of the reactor are heated or warmed to operating temperature. In some cases, the cultivated organisms generate so much waste heat through their metabolism that from a certain cell concentration only the cooling circuit is active. A heat exchanger can be integrated into this circuit or the energy-carrying medium is fed in directly. In most cases, only the double container wall and, in rare cases, built-in cooling registers , are available as heat exchange surfaces to the reaction chamber .

Oxygen content

Two fully automated steel fermenters from Heinrich Frings .

Fermentation approaches can, depending on the organism and product, be carried out aerobically (in an oxygen-containing atmosphere) or anaerobically (oxygen-free). Oxygen is poorly soluble in water, so that a sufficient supply of aerobic approaches is difficult. The oxygen solubility in a fermentation medium with a temperature of 37 ° C is, for example, 3-5 mg / L. The partial pressure of oxygen can be regulated by various methods:

Change in gas flow rate,
Change of the stirrer speed,
Change of the stirring tool geometry,
Change in gas mixture composition,
Change in head pressure (this also increases the solubility of other gases, for example carbon dioxide).

However, if gas is blown in too much or the stirrer speed is too high, the undesirable foam formation is also increased.

In the case of obligatory anaerobic organisms, however, oxygen supply should be avoided, as it can be toxic. In the case of anaerobic approaches with facultatively anaerobic organisms, an oxygen supply would enable undesired aerobic reactions which could reduce the process yield.

PH value

The cultivated organisms usually have a limited pH tolerance range with a pH optimum. The pH value can be controlled with pumps automatically coupled to a pH sensor, which can be used to acidify, for example, phosphoric acid (H ₃ PO ₄ ), hydrochloric acid (HCl) or to increase the pH value, for example sodium hydroxide (NaOH). pump into the bioreactor. In certain cases, the pH can also be achieved via the rate of substrate feeding.

Homogenization

Most bioreactors have a stirring device, such as. B. a stirrer or a gas injection through which the medium is circulated. This ensures a homogeneous setting of various parameters in the entire reactor and thus a more uniform process flow.

Foaming

The development of foam due to stirring is often problematic , which can clog the exhaust air filter and mechanically stress the cultivated cells. Chemical defoamers (antifoam agents) work by reducing surface tension . The negative impact on the gas transport and the poor separability from the reaction solution during downstream processing (product preparation).

Mechanical defoamers such as foam destroyers break up the foam, but do not remove the foam-forming factors such as e.g. B. dead cells. With foam separators, the foam is diverted and liquefied again and can then be pumped out.

Continuous or batch operation

When operating a fermenter, a distinction can be made between:

Batch operation: filling the reactor, no adding or removing during the fermentation process, simple control, contamination unlikely
Fed-batch operation: similar to batch operation; however z. B. Adding substrate during the process, as an initially high substrate concentration can be inhibiting
Continuous operation in the Chemostat bioreactor : uninterrupted operation through substrate addition and product removal, complex control, contamination is problematic, but expensive and time-consuming downstream processes can also be carried out continuously and thus optimally utilized

In research, batch fermentations are more likely to be carried out, while in larger production plants the establishment of continuous operation can make economic sense.

Reactors

Reactor types

The operating parameters to be observed for the organisms used or for technical, organizational and other reasons are very different. A corresponding bioreactor must therefore be designed for the respective use or a type of reactor can be used in which the various parameters can be regulated in a wide window so that it can be used for various purposes. A common type is the gasable stirred tank reactor in different versions (material, size, etc.).

Differentiation according to stirring technology

In every bioreactor there are three phases: solid ( biomass ), liquid (nutrient medium) and gaseous (e.g. air , oxygen, carbon dioxide , nitrogen ). In the bioreactor, their distribution is kept homogeneous with various measures:

Movable mechanical components (agitators): e.g. B. in a stirred tank reactor
external pump circuit: the liquid is circulated by a pump, e.g. B. Free jet reactor
Blowing in gas: the gas phase is blown into the liquid part e.g. B. Airlift reactor or bubble column reactor

If these reactor forms are provided with guide tubes , the following reactor types result:

Propeller loop reactor (a reactor in which energy is introduced by an axially downwardly conveying agitator and which is provided with a guide tube)
Jet loop reactor (a free jet reactor with a guide tube)
Mammoth loop reactor (an airlift reactor or a bubble column reactor with a draft tube)

Differentiation according to structure

A further distinction is possible based on the type of reactor structure:

Stirred tank reactor : common type; the liquid phase is circulated with a stirrer; fumigation takes place if necessary

Fixed bed reactor : the reactor is filled with a solid, porous matrix on which the organisms (or enzymes) can be immobilized; The organisms remain in the reactor instead of being washed out with the medium, so that the growth of the organisms is a less limiting factor (or the need for enzymes is reduced)

Trickling current reactor (trickling filter): a fixed bed is sprinkled with liquid (e.g. wastewater to be clarified); the degrading organisms sit on the surface; The air (oxygen) required for the decomposition is introduced in countercurrent

Photobioreactor ( algae reactor , hydrogen bioreactor ): for the cultivation of photosynthetic organisms (algae, plants (cells)); the reactor is made of glass so that the necessary light reaches the organisms; The use of light is optimized by plate or tubular reactors (see below: tubular reactor)

Tubular reactor : in tubular reactors, a plug flow can arise which z. B. is used in certain fermenters in biogas plants ; already degraded and fresh material ( substrate ) is not completely mixed, which can have various advantages

Membrane bioreactor : a reactor in which (depending on the application) the reaction product, the biomass or the purified water can be permanently separated using membranes . Applications for this are wastewater treatment (MBR), the production of lactic acid and pharmaceutical products.

Several stirred tank reactors connected in series form a cascade reactor ('stirred tank cascade '). In research and process development in particular, parallel bioreactor systems consisting of four, eight or sixteen reactors are increasingly being used.

In research, small stirred tank reactors or often also Erlenmeyer flasks are used as laboratory fermenters , which are attached to a so-called shaker to stir the medium.

In the past, solids bioreactors dominated in some areas because of their simpler process control . Liquid cultivation, also known as submerged fermentation , was difficult to control, but dominates today because of various advantages, such as better options for oxygen supply through stirring and aeration.

Reusable and disposable reactors

Most bioreactors are made of metal ( stainless steel ) or glass. This allows easy cleaning and sterilization and thus multiple use.

In animal cell culture technology, on the other hand, disposable bioreactors in the form of pre-sterilized disposable bags are increasingly being used. These consist of composite film . The single-use bags avoid the costly cleaning and sterilization processes, which in particular in the production of biological preparations leads to considerably shorter set-up times and thus to cost savings. Most single-use bioreactors are not stirred tank reactors. Instead, a rocking device takes over the circulation.

Applications

Digestion tower

Fermenter of a biogas plant

Sewage treatment plants

Very large bioreactors can be found in sewage treatment plants with biological process stages. In the activated sludge process , an aerobic step takes place in which the dissolved compounds of microorganisms are bound in the form of the biomass formed . This biomass can ultimately be fermented in the digestion tower to form methane-rich sewage gas (a digester gas ) . Another aerobic process is the trickling filter .

Biogas plants

The bioreactors of biogas plants are usually referred to as fermenters. The biomass used is broken down into biogas and digestate in an anaerobic process with several steps ( hydrolysis , acidogenesis , acetogenesis and methanogenesis ) . The containers are hermetically sealed and have an agitator and various measuring, control and regulation systems (MSR) for process control.

Breweries and wineries

Bioreactors are also needed in breweries or wineries . B. be referred to as fermentation tanks. The microorganisms used here are yeasts, which convert the sugar from the mash or grape juice into alcohol and carbon dioxide (CO ₂ ).

Pharmaceutical and cosmetic industries

The most valuable products manufactured in bioreactors are medical-pharmacological products such as erythropoietin (EPO), which has become known as a doping agent, or modern insulins . Since a significantly higher purity standard is set for medication than for food, particularly strict regulations apply here with the Good Manufacturing Practice guidelines. All operating parameters of the bioreactor must be kept within narrow limits, and even the smallest deviations mean that the entire batch may not be put into circulation. In order to be able to rule out as many imponderables as possible, fully biological nutrient media are rarely used in these processes, but an optimized, synthetic mixture of the required nutrients. This prevents the product quality from fluctuating as a result of fluctuations in the substrate quality. Depending on the desired product, different, mostly genetically modified, microorganisms are used in the pharmaceutical industry.

Modeling

The processes in bioreactors can be described by the reaction kinetics , whereby the special features of biological processes must be taken into account when modeling (e.g. Michaelis-Menten theory , Monod kinetics , enzyme kinetics , enzyme inhibition, etc.).

Individual evidence

^ Eva L. Decker, Ralf Reski : Current achievements in the production of complex biopharmaceuticals with moss bioreactor. In: Bioprocess and Biosystems Engineering , Volume 31, 2008, pp. 3-9. doi: 10.1007 / s00449-007-0151-y
↑ ^a ^b ^c ^d ^e ^f ^g Garabed Antranikian: Applied Microbiology , 1st edition, Springer-Verlag Berlin Heidelberg 2006, ISBN 3-540-24083-7 .
↑ Peter Czermak: Process intensification and efficient product manufacture with membranes. TH Mittelhessen , 2012, accessed on April 13, 2017 (PDF; 189 kB).
^ M. Barbaroux, A. Sette: Properties of Materials Used in Single-Use Flexible Containers: Requirements and Analysis . In: BioPharm International . 11, 2006.

literature

Winfried Storhas: Bioreactors and peripheral equipment: A guide for university education, for manufacturers and users . 1st edition. Springer Verlag, Heidelberg 1994, ISBN 3-540-67054-8 .
Ludwig Hasler, Rudolf Butz, Ueli Hepp: Transparency, Form and Function, Fermenter Construction - Art. 1st edition. Forest: DreiPunktVerlag, 2006. ISBN 978-3-905409-10-9 .
Karl Schwister (Hrsg.): Pocket book of the process engineering. 3rd edition, Fachbuchverlag Leipzig, Munich 2007. ISBN 978-3-446-41058-9 .
Garabed Antranikian : Applied Microbiology , 1st edition, Springer-Verlag Berlin Heidelberg 2006, ISBN 3-540-24083-7
Horst Chmiel (2006): Munich, Spektrum Akademischer Verlag, Bioprozesstechnik , 2nd edition ISBN 3-8274-1607-8

[1] Eva L. Decker, Ralf Reski : Current achievements in the production of complex biopharmaceuticals with moss bioreactor. In: Bioprocess and Biosystems Engineering , Volume 31, 2008, pp. 3-9. doi: 10.1007 / s00449-007-0151-y

[Garabed-2] ↑ ^a ^b ^c ^d ^e ^f ^g Garabed Antranikian: Applied Microbiology , 1st edition, Springer-Verlag Berlin Heidelberg 2006, ISBN 3-540-24083-7 .

[3] Peter Czermak: Process intensification and efficient product manufacture with membranes. TH Mittelhessen , 2012, accessed on April 13, 2017 (PDF; 189 kB).

[Barbaroux-4] M. Barbaroux, A. Sette: Properties of Materials Used in Single-Use Flexible Containers: Requirements and Analysis . In: BioPharm International . 11, 2006.