Cascade system for the fermentation of biomass

Abstract

The present invention relates to a cascade system for the fermentation of biomass, comprising a first pre-culture vessel, an optional second pre-culture vessel and a main-culture vessel, which are connected or can be connected to one another in this sequence by way of controllable valve couplings such that the content of one vessel can be transferred partially or completely into the next vessel, each vessel having an agitating, dispersing and / or cutting device, an inlet for the supply of nutrient medium, a pressure-equalizing valve and an inlet for compressed gas. The present invention also relates to a method for the fermentation of biomass, comprising (a) the provision of a starter culture, (b) the contamination-free transfer of the starter culture into a first pre-culture vessel, (c) the fermentation of biomass in the first pre-culture vessel, initiated by the starter culture, to obtain a first pre-culture, (d) the partial or complete transfer of the first pre-culture from the first pre-culture vessel into a main-culture vessel and (e) the fermentation of further biomass in the main-culture vessel to obtain a fermented biomass.

Description

[0001] Cascade system for biomass fermentation

[0002] The present invention relates to a cascade system for the fermentation of biomass, comprising a first pre-culture tank, an optional second pre-culture tank and a main culture tank, which are connected or connectable to one another in this order via controllable valve couplings such that the contents of one tank can be partially or completely transferred to the next tank, wherein each tank has a stirring, dispersing and / or cutting device, an inlet for supplying nutrient medium, a pressure equalization valve and an inlet for compressed gas.The present invention further relates to a method for fermenting biomass, comprising (a) providing a starter culture, (b) transferring the starter culture in a contamination-free manner into a first pre-culture container, (c) fermenting biomass in the first pre-culture container, which is initiated by the starter culture, in order to obtain a first pre-culture container, (d) partially or completely transferring the first pre-culture from the first pre-culture container into a main culture container and (e) fermenting further biomass in the main culture container in order to obtain a fermented biomass.

[0003] The large-scale production of fungal biomass (also known as mycelium) by fermentation is becoming increasingly important, as it is increasingly used as a food and, in particular, as a meat substitute.

[0004] To cultivate cells in large volumes, a fermentation reactor or vessel of suitable size for cultivating fungal biomass is usually inoculated using a so-called starter culture that has been incubated for several days in a separate, smaller container, e.g., a shake flask. From a process perspective, it has become established to first create a preculture or a series of increasingly larger precultures from the starter culture, before then inoculating the actual fermenter with such a preculture. This sequence of cultivation steps also makes it possible to revitalize the microorganism from a static starter culture (e.g., cryoculture), supply it with fresh nutrients with each passage into a subsequent preculture container, and ultimately cultivate sufficient cells to inoculate the main culture with abundant vital biomass.

[0005] Accordingly, conventional biomass fermentation is based on the manual inoculation of successive cultures. First, a small amount (i.e., the starter culture) of the microorganism, for example, a fungus, is usually taken from a frozen culture or an agar plate and transferred to a small volume of nutrient medium. This first preculture serves to revitalize the microorganism and is usually transferred partially or completely, and if necessary after a manual grinding step (e.g., with a disperser), to a second preculture. The second preculture serves primarily to propagate the biomass in a smaller volume than in the main culture.Typically, the second pre-culture is also crushed and used to start the main culture with as many vital cells as possible, which then, with the large amount of inoculum, quickly produces biomass and can be harvested after a relatively short time.

[0006] In this way, starting with an inoculum with a comparatively small number of cells, a large volume of culture medium with a high cell division rate can ultimately be cultivated. This accelerates the entire process and makes it more economical, as the technically and energy-intensive operation of the main culture vessel is designed as efficiently as possible.

[0007] Every transfer of a culture to the next reactor requires human intervention and thus carries the risk of contamination with an undesirable microorganism. Such contamination would jeopardize the bioprocess in the fermentation vessel. To best prevent contamination, an environment as sterile as possible, such as a sterile workbench, is required, as well as microbiologically trained personnel who typically wear personal protective equipment while working to prevent contamination.

[0008] In addition, the cultures usually need to be mechanically homogenized (either during cultivation or before transferring to the next fermentation vessel) to increase the vigor of the mycelium or other microorganisms by exposing new growth poles. This step is usually performed manually and requires opening the culture vessel, which further increases the risk of contamination. Therefore, a sterile environment and standardized workflows are necessary to prevent contamination of the culture with an undesirable microorganism.

[0009] This makes fermentation (not only using fungal cultures, but equally using bacterial, yeast, or other cell cultures) a very labor- and control-intensive process. Fermentation is labor-intensive because multiple vessels filled with medium must be inoculated and cultured with the desired microorganism in a sterile environment. The work involved in preparing and processing these cultures must be carried out on sterile benches using microbiological instruments (pipettes, sterile inoculation loops, spatulas, autoclaves, incubators, dispersing rods, etc.), which are complex to operate, clean, and maintain. Starter cultures must be prepared individually for each batch because they cannot be stored as liquid cultures for extended periods due to their decreasing viability.The process also requires intensive controls to ensure freedom from contamination at all steps. As explained above, the conventional production of starter, pre-culture, and main cultures, as well as their incubation and transfer, poses a microbiologic risk, as contamination with undesirable microorganisms exists at almost every process step, which would jeopardize the process's efficiency. Therefore, samples must be taken at various times to check for contamination. These samples are plated on suitable media and incubated. Furthermore, the samples are checked for purity using light microscopy and, if necessary, molecular biology (e.g., ITS sequencing). All of these procedures are necessary to ensure the purity of the cultures in this contamination-prone system.

[0010] The effort required for such a conventional fermentation process is clearly demonstrated, for example, by the publication by J. Alcantara et al. (Fermentation 2017, 3(3), 30).

[0011] Against this background, the present invention is based on the object of providing a system for the fermentation of biomass and a corresponding method, the use of which reduces the risk of contamination with an undesirable microorganism and reduces the labor and control effort in order to achieve an increase in the efficiency of the fermentation process.

[0012] This object is achieved by the embodiments characterized in the claims.

[0013] According to the invention, in particular, a cascade system for the fermentation of biomass is provided, comprising a first pre-culture container, an optional second pre-culture container and a main culture container, which are connected or connectable to one another in this order via controllable valve couplings in such a way that the contents of one container can be partially or completely transferred to the next container, wherein each container has a stirring, dispersing and / or cutting device, an inlet for supplying nutrient medium, a pressure equalization valve and an inlet for compressed gas.

[0014] In the context of the present invention, a cascade system is understood to mean a series of fermentation vessels or reactors that are or can be connected to one another in such a way that the contents of one vessel can be partially or completely transferred to the next vessel.

[0015] The cascade system according to the invention comprises a first preculture container, which serves to cultivate a first preculture. The cascade system according to the invention further comprises an optional second preculture container, which can be used to cultivate a second preculture. According to a further embodiment of the present invention, the cascade system also comprises a third preculture container, which serves to cultivate a third preculture. Additional preculture containers are also possible if required. The preculture containers can independently have any suitable size, for example, with volumes in the range of 10L to 500L. The volume of the first preculture container and the volume of the second preculture container are preferably in the range of 20L to 50L.It is further preferred that the volume of the optional second pre-culture container is larger than that of the first pre-culture container and that any further pre-culture containers are even larger than the second pre-culture container.

[0016] In a preferred embodiment, the cascade system according to the invention further comprises a starter culture container, which is arranged in the cascade upstream of the first pre-culture container and is connected or connectable to the first pre-culture container via a sterile coupling. The starter culture container serves to provide a starter culture, which can then be transferred to the first pre-culture container for the production of a first pre-culture. The sterile coupling is explained in more detail below. The starter culture container can have any suitable size, for example with volumes in the range of 1 L to 50 L. The volume of the optional starter culture container is preferably in the range of 5 L to 10 L. It is particularly preferred that the volume of the starter culture container is smaller than that of the pre-culture and main culture containers following in the cascade.

[0017] Finally, the cascade system according to the invention comprises a main culture container, which serves to cultivate the main culture. This container can also have any suitable size, for example, with volumes in the range of 1,000 L to 50,000 L. The volume of the main culture container is preferably in the range of 5,000 L to 20,000 L, particularly preferably in the range of 7,000 L to 10,000 L. In a preferred embodiment of the present invention, the volume of the main culture container is in the range between 1,000 L and 50,000 L, and the volume of the first and / or optional second preculture container is in the range between 10 L and 500 L.

[0018] According to the invention, the pre-culture and main culture containers are or can be connected to one another via controllable valve couplings in such a way that the contents of one container can be partially or completely transferred to the next container. Starting with the first pre-culture container, the containers are connected or can be connected to the main culture container via the second or possible further pre-culture containers. Any suitable valve couplings can be used for this purpose. These are known to the person skilled in the art. The controllable valve couplings are preferably controlled via a software-based process control system. Typically, the size of the culture increases over the course of the cascade, i.e. the starter culture represents a comparatively small culture and the first pre-culture is already larger in comparison. The optional second pre-culture and any further pre-cultures are then preferably also larger than the respective preceding pre-culture.The main culture serves to produce the fermented biomass on the largest possible scale and is therefore usually the largest of the cultures. Therefore, it is preferred that the volume of the containers in the cascade increases from the optional starter culture container via the first pre-culture container via the optional second pre-culture container (and optionally further pre-culture containers) to the main culture container.

[0019] Each of the aforementioned containers preferably comprises a housing and a housing lid. The housing and lid of each container can independently be made of any suitable material. Preferably, the housing and lid are made of heat- and pressure-resistant materials so that they can easily withstand the heat sterilization and autoclaving conditions typical for fermentation. In a preferred embodiment of the present invention, the housing and / or the lid are made of stainless steel. Each container can independently also have any suitable shape. For example, the containers can have a substantially cylindrical shape, as is typical for fermentation reactors.

[0020] Other shapes, for example with a rectangular, square or oval cross-section, are also possible.

[0021] The housing and the lid are connected or connectable to one another. According to a preferred embodiment, the containers can be aseptically closed by the lid. In the context of the present invention, an aseptically sealable lid is understood to mean that it can be closed in a contamination-proof and gas-tight manner, so that no foreign germs can penetrate the containers when closed. The lid can be opened for filling and / or cleaning. In a preferred embodiment of the present invention, the lid is connected or can be connected to the housing by means of a screw cap, by means of a twist lock, by means of a compression fitting via clamps or by means of one or more clamping rings. The screw cap can be, for example, a dairy pipe fitting made of stainless steel. A compression fitting via clamps is particularly preferred.

[0022] Each of the pre-culture and main culture vessels of the cascade system according to the invention has a stirring, dispersing, and / or cutting device. This serves in particular to crush the solid portion of the vessel contents and / or to stir the vessel contents. In this way, the vigor of the cell culture (for example, the mycelium) can be increased by exposing new growth poles. Furthermore, comminution facilitates the subsequent transfer of the vessel contents to another fermentation vessel. The stirring, dispersing, and / or cutting device can be any device suitable for comminuting the solid portion of the vessel contents and / or for stirring the vessel contents. Preferably, the stirring, dispersing, and / or cutting device is selected from a disperser, a chopper, and / or an agitator. The stirring, dispersing, and / or cutting device can be arranged at any suitable position in the vessel.For example, it can be attached to the inside of the lid and protrude into the container from above. Alternatively, it is also possible for the stirring, dispersing, and / or cutting device to be located at the bottom of the housing. In this case, it preferably protrudes into the container from below. Other configurations are also possible. The container can, for example, have a flanged pipe system on the side through which the container contents can be pumped by means of a pump and in which the stirring, dispersing, and / or cutting device is also located.

[0023] Each of the pre-culture and main culture containers of the cascade system according to the invention further has an inlet for supplying nutrient medium. Components other than the nutrient medium can be added via this inlet if required. The inlet can be any suitable opening on the container through which nutrient medium and optionally also further components can be supplied. The inlet can, for example, be the aforementioned housing cover. However, it is also possible for there to be a further opening on the housing or housing cover through which the nutrient medium can be supplied. According to a preferred embodiment of the present invention, the controllable valve coupling described above on the respective container represents the inlet for supplying nutrient medium. According to another preferred embodiment of the present invention, the inlet for supplying nutrient medium is a sterile coupling.In the context of the present invention, a sterile coupling is understood to be a connecting coupling for pipes or hoses that can be sterilized. This can be made, for example, from stainless steel, titanium, alloys of nickel, molybdenum, chromium and iron, and combinations thereof. In a preferred embodiment of the present invention, the sterile coupling is free of dead spaces. In a further preferred embodiment, the sterile coupling is suitable for in-line sterilization with steam (SIP - "Sterilization in Place"). Most preferably, the sterile coupling is a flat-sealing sterile coupling. The sterile coupling is designed such that nutrient medium and, if necessary, other components can be supplied to the container via it and / or the container contents can be partially or essentially completely removed or transferred to another container without the container having to be opened.

[0024] Each of the pre-culture and main culture containers of the cascade system according to the invention further comprises a pressure equalization valve, which serves to prevent the buildup of excess pressure in the container during sterilization or fermentation. Like the inlet for supplying nutrient medium, the pressure equalization valve can be located at any suitable location on the container, for example, on the lid or on the housing. If the pressure equalization valve is located on the housing, it should preferably be located above the maximum fill level of the container, for example, in the upper third of the housing. According to a preferred embodiment, the pressure equalization valve is a spring-loaded check valve.

[0025] Each of the containers of the cascade system according to the invention further comprises a compressed gas inlet for supplying compressed gas, which serves to aerate (preferably supply oxygen) the container contents and / or to stir the container contents. The compressed gas inlet can be arranged at any suitable location on the container, for example on the lid or on the housing. Preferably, however, it is arranged on the housing and there particularly preferably below the maximum fill level of the container, for example in the lower third of the housing. In a preferred embodiment of the present invention, the compressed gas inlet is a compressed air inlet valve.

[0026] Each of the containers can comprise further optional components. For example, the container can further comprise a heating device configured to sterilize the container contents and / or to provide the required temperatures for fermentation. This heating device can be arranged, for example, at the bottom of the housing and / or in the shell or side walls of the housing (possibly in the form of a double-walled container).

[0027] In a further preferred embodiment of the present invention, the cascade system further comprises a software-based process control system for controlling and monitoring the fermentation and the transfer of the contents of one container to the next container, or is connectable to the next container. For this purpose, the software-based process control system can be used, in particular, to control the controllable valve couplings. It is further particularly preferred that at least one container has a sensor for measuring the growth of the culture in the container, which sensor is connected or connectable to the software-based process control system. Preferably, all containers comprise a corresponding sensor. Suitable sensors include, for example, temperature, pH, and dissolved oxygen sensors. Photometric sensors, with which the penetration of a light signal through the culture can be measured, are also suitable.These sensors make it possible to record the progress of the fermentation and, accordingly, initiate the comminution of the respective container contents and the transfer of one container content to the next (e.g., from the first pre-culture container to the main culture container). The software-based process control system allows all of this to be automated and carried out at the most optimal time (particularly with regard to the control of the valves that release the contents of one container into the next), so that no active monitoring of the process by laboratory personnel is necessary. For example, the first pre-culture can be released from the first pre-culture container into the next container after approximately 4 to 6 days, the optional second pre-culture can be released from the second pre-culture container into the next container after approximately 3 to 5 days, and the main culture can be released, for example, after approximately 5 to 10 days.The software-based process control system can also control the mixing of the biomass in the individual containers via the inflow of compressed air and / or via the agitators. For example, the mixing speed and air input into the system can be increased with increasing cell density / biomass. This can be controlled, for example, based on the dissolved oxygen concentration or viscosity. For example, a specific oxygen value can be specified as a setpoint.

[0028] In a preferred embodiment of the cascade system according to the invention, at least one of the containers is connected or connectable to a cleaning and / or sanitizing system. Particularly preferably, all containers are connected or connectable to a central cleaning and / or sanitizing system. Such systems are known to those skilled in the art. Furthermore, it is preferred that at least one container inlet for supplying nutrient medium has a steam sterilization device. Particularly preferably, all containers have a steam sterilization device at the container inlet. In this way, the cleaning or sanitizing of the container(s) and the corresponding inlets can also be automated, without the need for manual cleaning or sanitizing by laboratory personnel.

[0029] The cascade-like design of the inventive cascade system for biomass fermentation and the connection of the individual containers via controllable valves eliminates the need for human intervention during the incubation of the precultures. Therefore, a process operated with this system does not require microbiologically trained personnel. Furthermore, the modular design of the individual containers allows for flexible expansion of the system according to the individual requirements of a given bioprocess. Furthermore, the optional integration of all containers into a central cleaning and sanitization system facilitates cleaning and sanitization of the system and largely eliminates the need for human intervention. Manual cleaning and sterilization of individual vessels and instruments is thus eliminated.The installation of a comminution system in each container also enables fully automated comminution of the biomass before transfer to the next container, without the need to open the container. This reduces labor and the risk of contamination with foreign germs. In a preferred embodiment, the cascade system can be inoculated without contamination using a starter culture, which can preferably be connected to the first pre-culture container using a dead-space-free sterile coupling. This reduces labor, instrumentation requirements (regarding a sterile laboratory), and the risk of contamination with foreign germs. The preferably computer-assisted process control optimizes the process flow and ensures the transition from one container to the next during the optimal growth phase of the culture. Furthermore, the comminution of the biomass is optimally controlled.

[0030] The present invention further relates to a method for the fermentation of biomass, comprising (a) providing a starter culture, (b) transferring the starter culture in a contamination-free manner, preferably via a sterile coupling, into a first pre-culture container, (c) fermenting biomass in the first pre-culture container, which is initiated by the starter culture, in order to obtain a first pre-culture, (d) partially or completely transferring the first pre-culture from the first pre-culture container into a main culture container and (e) fermenting further biomass in the main culture container in order to obtain a fermented biomass.

[0031] In step (a) of the process according to the invention, a starter culture is first provided. According to the invention, any suitable starter culture for the particular envisaged fermentation can be used. For example, these can be starter cultures for fermentation using bacterial, fungal, or other biological cell cultures. The starter culture can also contain enzymes (ferments) that carry out the fermentation as part of their enzyme-catalyzed metabolism. The starter culture preferably contains fungal biomass (mycelium). The fermentation is carried out using fungal cultures. Accordingly, the fermented biomass—as a product of the process according to the invention—preferably also contains fungal biomass (mycelium). However, the present invention can also be used for fermentations using yeasts, for example, in connection with precision fermentation processes.Preferably, the starter culture is provided in the starter culture container described above. In step (b) of the method according to the invention, the starter culture is transferred in a contamination-free manner into a first pre-culture container. This transfer preferably takes place from a starter culture container that has a sterile coupling. The first pre-culture container is preferably the first pre-culture container described above. Corresponding sterile couplings have also already been described above.

[0032] In step (c) of the process according to the invention, biomass is fermented in the first preculture container, initiated by the starter culture, to obtain a first preculture. Any biomass suitable as a nutrient medium for cultivating microorganisms can be used for this purpose. Suitable nutrient media are known to those skilled in the art and can be selected depending on the intended fermentation product. Generally speaking, a suitable nutrient medium consists of at least one carbohydrate and nitrogen source, which serve as a substrate, for example, for the fungal cells. The nutrient medium can be, for example, a starch- and / or protein-containing flour or an artificial nutrient medium. For example, it can be any N- and C-containing agro-industrial side stream (starch-containing fractions from protein isolation (e.g., from legumes), bran, etc.).Additionally, the medium can optionally contain a gelling agent such as starch, agar agar, agarose, pectin, or gelatin to enable hardening or gelling upon heat treatment. A starchy fraction from peas, a byproduct of protein isolation, is particularly preferably used as the biomass. This fraction is sufficient in terms of C and N sources and gels thanks to the starch. Heat treatment in this context refers to treatment at a temperature of at least 72°C. Preferably, a temperature of at least 95°C is used for the heat treatment, particularly preferably a temperature in the range of 105°C to 125°C.

[0033] The biomass is fermented in a conventional manner known to those skilled in the art. The duration of the fermentation can be selected as appropriate. According to a preferred embodiment of the present invention, the fermentation duration in step (c) is 2 to 14 days, and preferably 5 to 10 days. Furthermore, compressed air can be supplied during fermentation. This serves for aeration (preferably the oxygen supply) and / or stirring of the container contents. In a preferred embodiment, the contents of the first preculture container are comminuted or homogenized at least once during fermentation using a dispersing and / or cutting device for a duration of at least 10 seconds.This makes it possible to mechanically break up unwanted pellets that form during fermentation (e.g., mycelium pellets) during ongoing mycelium cultivation without human intervention and without the risk of introducing unwanted foreign germs. This makes it possible to expose new growth poles during the ongoing process, rejuvenate the mycelium, and increase the overall yield. According to the invention, the container contents are circulated in a sterile state and simultaneously crushed to break up and crush the pellets. Individual fragments (e.g., mycelium fragments) are released into the surrounding medium. These can re-branch and thus grow at multiple poles. Since the crushing of the pellets neither requires interruption of the fermentation process nor opening of the container, there is no increased risk of contamination.

[0034] In step (d) of the method according to the invention, the first preculture is partially or completely transferred from the first preculture container into a main culture container. This is preferably the main culture container described above. As described above, this main culture container is preferably larger than the first preculture container.

[0035] Finally, in step (e), additional biomass is fermented in the main culture vessel to obtain a fermented biomass. The same explanations apply to this fermentation step as previously described for the fermentation step in the first pre-culture vessel, except that a larger amount of main culture is produced compared to the pre-culture. The fermented biomass preferably contains fungal biomass.

[0036] In a preferred embodiment, the process according to the invention is carried out in the cascade system according to the invention described above. According to a further preferred embodiment, the first preculture is first transferred to a second preculture container, in which further biomass is fermented and then partially or completely transferred to the main culture container. Preferably, the process further comprises comminuting and / or stirring the container contents during at least one fermentation step. Likewise, it is preferred that the process further comprises the step of aerating the container contents with air during at least one fermentation step.

[0037] The invention will now be described using an embodiment with reference to a schematic representation of a container according to the invention for the fermentation of biomass.

[0038] Figure 1 shows a cascade system comprising a starter culture container 1 with a sterile coupling 2. The starter culture container 1 is connected to a first pre-culture container 3 via the sterile coupling 2. The first pre-culture container 3 is in turn connected to a second pre-culture container 5 via a controllable valve coupling 4. This second pre-culture container 5 is connected to a main culture container 7 via a further controllable valve coupling 6. Each of the containers comprises a housing and a housing lid (which also serves as an inlet for supplying nutrient medium). The pre-culture containers 3 and 5, as well as the main culture container 7, further comprise a dispersing device, a pressure equalization valve, and an inlet for compressed gas (not shown).

[0039] List of reference symbols

[0040] 1 starter culture container

[0041] 2 sterile coupling

[0042] 3 First pre-culture container

[0043] 4 Controllable valve coupling

[0044] 5 Second pre-culture container

[0045] 6 Controllable valve coupling

[0046] 7 main culture containers

Claims

Patent claims 1. Cascade system for the fermentation of biomass, comprising - a first pre-culture container, - an optional second pre-culture container and - a main culture container, which are connected or connectable to each other in this order via controllable valve couplings in such a way that the contents of one container can be transferred partially or completely into the next container, each container - a stirring, dispersing and / or cutting device, - an inlet for the supply of nutrient medium, - a pressure equalization valve and - has an inlet for compressed gas.

2. Cascade system according to claim 1, which further comprises a starter culture container which is arranged in the cascade in front of the first pre-culture container and is connected or connectable to the first pre-culture container via a sterile coupling.

3. Cascade system according to claim 1 or 2, wherein the volume of the containers in the cascade increases from the optional starter culture container via the first pre-culture container via the optional second pre-culture container to the main culture container.

4. Cascade system according to one of claims 1 to 3, wherein the volume of the main culture container is in the range between 1,000L and 50,000L and the volume of the first and / or optional second pre-culture container is in the range between 10L and 500L.

5. Cascade system according to one of claims 1 to 4, which further comprises a software-based process control system for controlling and monitoring the fermentation and the transfer of the contents of one container into the next container or is connectable thereto.

6. Cascade system according to claim 5, wherein at least one container has a sensor for measuring the growth of the culture in the container, which sensor is connected or connectable to the software-based process control system.

7. Cascade system according to one of claims 1 to 6, wherein at least one container is connected or connectable to a cleaning and / or sanitizing system.

8. Cascade system according to claim 7, wherein all containers are connected or connectable to a central cleaning and / or sanitizing system.

9. Cascade system according to one of claims 1 to 8, wherein at least one container inlet for supplying nutrient medium has a steam sterilization device.

10. Cascade system according to one of claims 1 to 9, wherein the controllable valve coupling on the respective container represents the inlet for supplying nutrient medium.

11. Cascade system according to one of claims 1 to 10, wherein the stirring, dispersing and / or cutting device is selected from a disperser, a chopper and / or an agitator.

12. A process for the fermentation of biomass, comprising (a) providing a starter culture, (b) the contamination-free transfer of the starter culture, preferably via a sterile coupling, into a first pre-culture container, (c) fermenting biomass in the first pre-culture container, which is initiated by the starter culture, to obtain a first pre-culture, (d) the partial or complete transfer of the first pre-culture from the first pre-culture container into a main culture container and (e) fermenting further biomass in the main culture vessel to obtain a fermented biomass.

13. The method according to claim 12, which is carried out in the cascade system according to one of claims 1 to 11.

14. The method according to claim 12 or 13, wherein the first preculture is first transferred to a second preculture container, in which further biomass is fermented and then partially or completely transferred to the main culture container.

15. The method according to any one of claims 12 to 14, which further comprises comminuting and / or stirring the container contents during at least one fermentation step.

16. A method according to any one of claims 12 to 15, which further comprises the step of aerating the container contents with air during at least one fermentation step.

17. The method according to claim 16, wherein the fermented biomass contains fungal biomass (mycelium).

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