Bioreactor for producing mycoprotein and method of operating such bioreactor

Abstract

A bioreactor (10) for producing a protein, such as a mycoprotein and / or mycelium fibers, is proposed. The bioreactor (10) comprises a reactor body (12) having a reactor wall (14), the reactor wall (14) confining a reactor volume for incubating an inoculum for producing the protein, the reactor wall (14) having a tapering bottom section (16); a bottom opening (18) provided in the reactor wall (14) at the tapering bottom section (16); and a cap (20) for covering the bottom opening (18), the cap (20) including a top face (50), and a first fluid channel (36) extending through the cap (20), wherein the first fluid channel (36) is configured for transferring fluid between an inside of the reactor body (12) and an outside of the reactor body (12), and wherein an exit opening (46) of the first fluid channel (36) is arranged flush with the top face (50) of the cap (20).

Description

[0001] New PCT application

[0002] Applicant: Pacificolabs Biolabs GmbH

[0003] Vossius Ref.: AJ3904 PCT BS

[0004] Bioreactor for Producing Mycoprotein and Method of Operating such Bioreactor

[0005] Field of the invention

[0006] The present invention relates to a bioreactor. More specifically, the present invention relates to a bioreactor for producing a protein, such as a mycoprotein, in particular mycelium or mycelium fibers, for use as food alternative or food analogue. The present invention further relates to a method of producing a protein, preferably in such a bioreactor.

[0007] Background of the invention

[0008] Food analogues are a promising alternative to traditional foods. Food analogues will become increasingly important in the future amid rising concerns on limited availability and / or sustainable production of traditional foods.

[0009] Recently, food analogues from fermented fungal mycelia, also known as mycelium, have attracted large interest. Mycelia can be used as "building material" for producing large amounts of alternative food such as meat, fish, cheese, etc. at a comparably low cost and in a scalable manner. Mycelium is typically produced from an inoculum incubated in a bioreactor. The inoculum is fed with a cultivation medium that includes nutrients for germinating and growing mycelia. When enough mycelium is produced, the mycelium may be harvested from the bioreactor and may be processed further to produce the desired food analogue.

[0010] One type of bioreactor that seems to be ideally set for growing mycelium is a so-called fermentation tank. A typical bioreactor used in advanced biotechnology is a stirred tank bioreactor (STR). Those tanks are widely used in the bioprocessing of several molecules. However, they are capex intensive and require intense mechanical mixing and cooling that in turn result in high energy demands. Furthermore, it has been found by the inventors that an STR with high agitation leads to high shear stress, which may be disadvantageous.

[0011] Fermentation tanks used in the brewery industry on the other hand are not suitable for advanced bioprocessing. In the brewery industry, inoculated substrate such as yeast, hop, etc. is filled into the tank, processed for a predetermined time and then harvested from the tank.

[0012] Fermentation tanks used in the brewery industry are known, for example, from EP 3 365 422 Al. The tank includes a cylindroconical tank body with a conically shaped lower tank region. The lower tank region includes a central opening. An aeration lance is inserted through the central opening. The aeration lance protrudes into the inside of the tank. The aeration lance is used for aerating and circulating the inoculated substrates during fermentation of beer.

[0013] Using an aeration lance may, however, be not ideal when it comes to a fungal submerged fermentation process to produce, for example, mycelium mycoprotein biomass.

[0014] It is thus an object of the present invention to propose a bioreactor which overcomes at least some drawbacks encountered in known fermenters. It is further an object of the present invention to propose a bioreactor which is better suited for production of proteins such as mycelium than known STRs, airlift bioreactors or brewery fermenters. It is further an object of the present invention to propose a method of producing proteins such as mycelium using such a bioreactor. It is further an object of the present invention to propose a low-cost and simple modification to known brewery fermenters such that those fermenters can be advantageously used in the production of mycoproteins, such as mycelium.

[0015] Solution to the problem

[0016] These and other objects, which become apparent upon reading the description, are solved by the subjectmatter of the independent claims. Further embodiments and developments are provided in the dependent claims.

[0017] According to a first aspect of the present invention, a bioreactor for producing a protein, such as a mycoprotein and / or mycelium fibers, is proposed. The bioreactor comprises a reactor body having a reactor wall, the reactor wall confining a reactor volume for incubating an inoculum for producing the protein, the reactor wall having a tapering bottom section; a bottom opening provided in the reactor wall at the tapering bottom section; and a cap for covering the bottom opening. The cap includes a top face and a first fluid channel extending through the cap, wherein the first fluid channel is configured for transferring fluid between an inside of the reactor body and an outside of the reactor body. Preferably, an exit opening of the first fluid channel is arranged flush with the top face of the cap.

[0018] The present invention is at least partially based on the idea that niches, steps or protrusions inside the reactor body may promote adhering and / or growing of mycelia in these regions. The idea of the present invention is to avoid any such niches, steps etc. by arranging the exit opening of the first fluid channel (aeration channel) flush with the top face of the cap. The inventors observed that niches, steps, etc. are preferred regions where mycelium may adhere and / or grow. Over time, this may result in clogging of the reactor. Furthermore, it is assumed that this may lead to fungal biofouling. The idea of the proposed reactor design is to prevent any unnecessary clogging by providing a smooth and flush top face of the cap. In addition, fungal biomats grown inside niches of the fermenter are particularly disadvantageous because they are not suitable for developing food alternatives. Without being bound by theory, it is believed that any parts which protrude into the reactor volume, such as the aeration lance known from EP 3 365 422 Al, may be disadvantageous when it comes to growing mycelia. It is believed that mycelia may adhere to the aeration lance of EP 3 365 422 Al, and / or may get tangled around the aeration lance. It is believed that this may lead to clogging of the reactor and / or clogging of at least a discharge line in the vicinity of the aeration lance. The reactor of EP 3 365422 Al is used for fermenting yeast, mash etc. The fermentation of yeast, mash etc. for production of beer produces a rather thin broth during fermentation. It is believed that such thin broths may not clog the reactor even when using a protruding aeration lance as taught by EP 3 365 422A1. The fermentation process to obtain fungal mycelia, on the other hand, results in a mycelium broth that is more viscous and / or thicker compared to known beer broths. In addition, it is also a physiological feature of mycelium to attach and form biomats or biofilms on surfaces. It is thus believed that any aeration lance or other protruding lines in connection with fermentation of fungal mycelia may enhance clogging of the reactor. The proposed reactor design, therefore, uses a cap with a flush and smooth top face. It is believed that by using such cap, clogging of the reactor during fermentation to obtain mycelia can be prevented or at least reduced.

[0019] For example, it has been surprisingly found by the inventors that a bioreactor in accordance with the present invention may allow to obtain substantially longer fibers of mycelium. Without wanting to be bound by theory, it is believed that such longer fibers of mycelium may be obtained in view of the absence of a high agitation and / or the absence of a mechanical mixing implement. It is also believed that avoiding tangling and / or fouling of fibers within the reactor may further help in obtaining such long fibers of mycelium and / or to preserve the quality of the fibers.

[0020] The present invention is also based on the idea that known brewery fermenters, which are currently primarily used for beer production, could be retrofitted by adding a flush aeration cap at the bottom of the fermenter. The inventors believe that brewery fermenters modified in this way may be advantageously used for production of mycoproteins, such as mycelium.

[0021] Preferably, the top face of the cap is planar, and / or is configured for providing a smooth transition to an inner surface of the reactor wall. The top face may be concave.

[0022] Preferably, in a region around the bottom opening, the top face of the cap and an inner surface of the reactor wall are arranged flush to one another.

[0023] Preferably, the reactor body comprises a cylindrical body section, wherein the cylindrical body section and / or the tapering bottom section have a main axis. Preferably, the first fluid channel has a first fluid channel axis, and the first fluid channel axis has an angled orientation to the main axis. Preferably, an angle between the main axis and the first fluid channel axis is in a range between 10° to 60°, more preferably between 20° to 30°, and / or an angle between the main axis and the first fluid channel axis is adapted for creating a fluid swirl within the reactor volume. A throughput of air or oxygen through the first fluid channel may be adapted by the pressure and / or flow rate of air / oxygen.

[0024] Preferably, the exit opening is configured for producing air and / or oxygen bubbles having a size, such as an average diameter, of at least 5 mm, preferably at least 8 mm, more preferably at least 10 mm, more preferably at least 20 mm, even more preferably at least 30 mm, and / or preferably wherein the air and / or oxygen bubbles have a size in a range between 10 mm and 40 mm, preferably between 10 mm and 30 mm, more preferably between 10 mm and 20 mm.

[0025] Without wanting to be bound by theory, the inventors have found that air and / or oxygen bubbles of 10 mm or more may be particularly beneficial for creating an effective air lift within the reactor volume.

[0026] Preferably, the first fluid channel and / or the exit opening includes a diameter of at least 10 mm, preferably at least 15 mm, more preferably at least 20 mm, more preferably at least 25 mm. The exit opening preferably includes a diameter in a range between 10 mm and 60 mm, preferably 15 mm and 60 mm, more preferably 20 mm and 60 mm, more preferably 25 mm and 60 mm. Preferably, the exit opening has a diameter of 50 mm or less.

[0027] Without wanting to be bound by theory, the inventors have found that a bubble size provided at the exit opening is, to a large extent, determined by a diameter of the first fluid channel and / or a diameter of the exit opening of the first fluid channel. The inventors have found that a diameter of the first fluid channel and / or the exit opening of at least 25 mm is advantageous for generating air and / or oxygen bubbles of 10 mm or more. Exit openings or fluid channels with smaller diameters tend to produce smaller bubbles, which have been found to be less effective in generating the desired air lift effect (internal recirculation / mixing) and, consequently, may provide inadequate stirring or mixing within the reactor. This, of course, depends somewhat on the viscosity of the medium in the reactor.

[0028] By ensuring that the exit opening or openings and / or first fluid channel are sufficiently large, the resulting air and / or oxygen bubbles are more likely to establish a robust stir and / or recirculation zone within the reactor volume. This enables efficient mixing and recirculation of the reactor contents. Thereby, a mechanical stirrer or other mixing devices within the reactor may not be required and can be omitted. This may be advantageous because such mechanical stirrer or mixing devices may be difficult to retrofit in existing reactors and / or may be detrimental for obtaining certain types of mycelia.

[0029] Preferably, the exit opening is delimited by an inner wall of the first fluid channel.

[0030] Preferably, a perimeter of the exit opening is defined by an inner wall of the first fluid channel.

[0031] Preferably, the first fluid channel includes a plurality of exit openings, each exit opening being arranged flush with the top face of the cap. In other words, multiple exit openings may be provided flush with the top face for introducing air / oxygen into the reactor volume. Preferably, the exit openings are configured for producing air and / or oxygen bubbles having a size of at least 5 mm, preferably at least 8 mm, more preferably at least 10 mm, more preferably at least 20 mm, even more preferably at least 30 mm, and / or preferably wherein the air and / or oxygen bubbles have a size in a range between 10 mm and 40 mm, preferably between 10 mm and 30 mm, more preferably between 10 mm and 20 mm.

[0032] Preferably, the exit openings are delimited by an inner wall of the first fluid channel.

[0033] Preferably, a perimeter of the exit openings is defined by an inner wall of the first fluid channel.

[0034] Preferably, the exit openings include a diameter of at least 10 mm, preferably at least 15 mm, more preferably at least 20 mm, more preferably at least 25 mm. The exit openings preferably include a diameter in a range between 10 mm and 60 mm, preferably 15 mm and 60 mm, more preferably 20 mm and 60 mm, more preferably 25 mm and 60 mm. Preferably, the exit openings have a diameter of 50 mm or less.

[0035] Preferably, the reactor volume is free of a stirring device, such as an impeller having a shaft and / or stirring blades. The inventors have found that such stirring device can have a negative effect on the size / length of mycelia grown in the bioreactor.

[0036] Preferably, the bioreactor is configured for producing mycelium fibers, such as a mycoprotein fibers and / or mycelium hyphae, having a minimum length of at least 0.4 mm, preferably at least 0.5 mm, more preferably at least 0.8 mm, more preferably at least 1 mm, more preferably at 1.5 mm, such as for example 1.97 mm.

[0037] Preferably, at least 60%, preferably at least 80%, more preferably at least 90%, more preferably at least 95% of the produced mycelium fibers, such as a mycoprotein fibers and / or mycelium hyphae, have a length of at least 0.4 mm, preferably at least 0.5 mm, more preferably at least 0.8 mm, more preferably at least 1 mm, more preferably at least 1.5 mm, such as for example 1.97 mm.

[0038] Preferably, the bioreactor is configured for producing mycelium fibers, such as a mycoprotein fibers and / or mycelium hyphae, having an average length of at least 2 mm, preferably at least 3 mm, preferably at least 4 mm. Preferably, the bioreactor is configured for producing mycelium fibers, such as mycoprotein fibers and / or hyphae, having a maximum length of at least 5 mm, preferably at least 8 mm, more preferably at least 10 mm.

[0039] The bioreactor may be configured for producing mycelium fibers, such as a mycoprotein fibers and / or hyphae, having a average length of 25 mm or less, preferably 20 mm or less, preferably 15 mm or less.

[0040] Preferably, the bioreactor is configured for producing mycelium fibers, such as mycoprotein fibers and / or hyphae, with a production rate of at least 10 g / L / d, preferably at least 15 g / L / d, more preferably at least 20 g / L / d, even more preferably at least 30 g / L / d, preferably wherein the produced mycelium fibers (in particular, the mycelium hyphae) have an average length of at least 2 mm, preferably at least 3 mm, preferably at least Preferably, the first fluid channel includes a main channel portion and a ring channel portion, and the ring channel portion fluidly connects the main channel portion with the plurality of exit openings. In other words, a ring channel portion may distribute the air / oxygen to the plurality of exit openings.

[0041] The ring channel portion may be a full or a partial ring (e.g., it may subtend an angle of at least 180°, at least 270° or 360°). As such, it may, but does not necessarily, form a closed ring.

[0042] The ring channel portion may extend around at least partially or fully around a second fluid channel for discharging protein. This second fluid channel is discussed in more detail hereinbelow.

[0043] Preferably, the exit openings are arranged in a predetermined pattern on the top face of the cap, preferably wherein the predetermined pattern is a circular pattern around an imaginary central point.

[0044] Preferably, the exit openings extend at exit opening axes that are oblique to the main axis, preferably wherein the exit opening axes are arranged or extend at an angle to the main axis which is in a range between 10° to 60°, more preferably between 20° to 30°, more preferably wherein the angle is configured to create a fluid swirl within the reactor volume. In other words, the exit openings may be in an angled configuration with the main axis of the reactor body.

[0045] Preferably, the exit opening axes, e.g. via their angled configuration, are configured such that the exiting air introduces a swirl in the reactor volume. The swirl may be used for mixing of a broth within the reactor body. In particular, it is believed that such swirl may provide for a mixing of a mycelium broth in the reactor wherein breakage of grown mycelia is minimized. It is believed that such mixing may allow to achieve longer mycelia during cultivation. It is believed that such longer mycelia may allow to produce a superior food analogue.

[0046] Preferably the first fluid channel is configured for aerating a mixture of inoculum and a cultivation medium in the reactor volume.

[0047] Preferably, the cap includes a second fluid channel that is separate from the first fluid channel, the second fluid channel extending through the cap and being configured for transferring fluid between the inside and the outside of the reactor body. An exit opening of the second fluid channel is preferably arranged flush with the top face of the cap.

[0048] Preferably, the second fluid channel is configured for discharging proteins and / or a broth including proteins, such as mycoproteins. Preferably, the second fluid channel has a diameter of at least 10 mm, more preferably at least 15 mm, and / or the second fluid channel has a diameter of 15 to 20 mm or larger.

[0049] Preferably, the cap includes less than 5 fluid channels, such as 4 or 3 fluid channels. Preferably the cap includes less than 5 exit openings. Preferably, the cap includes one or two first fluid channels configured for aeration and / or providing air bubbles to the reactor volume and one or two, preferably a single, second fluid channel configured for discharging proteins and / or a broth including proteins, such as mycoproteins.

[0050] Preferably, the cap includes a single, preferably exactly two, more preferably less than 5 first fluid channels for aeration and / or providing air bubbles to the reactor volume.

[0051] Preferably, the second fluid channel is parallel with a central or main axis of the reactor body.

[0052] Preferably, a diameter of the second fluid channel is larger than a diameter of the first fluid channel and / or an exit opening of the first fluid channel. For example, a diameter of the second fluid channel preferably is larger by a factor of 4, 6, 8, 10 or more than a diameter of the first fluid channel and / or an exit opening of the first fluid channel. The diameter of the second fluid channel may be adjusted such that no clogging occurs during production and harvesting of proteins.

[0053] Preferably, the exit openings of the first fluid channel are arranged in a predetermined pattern around the exit opening of the second fluid channel, and / or the exit openings of the first fluid channel are arranged in a circular pattern and / or in a concentric manner around the exit opening of the second fluid channel. For example, in embodiments with multiple exit openings for the first fluid channel (aeration channel), these multiple exit openings may be arranged concentric around the exit opening of the second fluid channel.

[0054] Preferably, the cap is removably connected to the bottom opening.

[0055] Preferably, the cap is screwed, or clamped onto and / or into the bottom opening.

[0056] The cap and the bottom opening may be connected to each other via a threading, a bayonet connection, or a clamping mechanism. For example, the cap body may include an outer thread adapted to an inner thread of the reactor wall at the bottom opening.

[0057] Preferably, the cap is sealingly fitted into the bottom opening. Preferably, the cap is removably fitted, preferably threadedly fitted, into the bottom opening.

[0058] Preferably, the cap includes a port or a coupling for coupling at least one pipe or line, preferably a plurality of pipes or lines, to the cap, preferably wherein the cap is configured for coupling at least one pipe or line using a dairy coupling. Such a dairy coupling may be a coupling in accordance with DIN 11851.

[0059] Preferably, the cap has one of a male or female connection port connected to the reactor wall for coupling the cap with the bottom opening of the reactor wall. Preferably, wherein the cap includes a tubular section connected to the one of a male or female connection port, wherein the first fluid channel is coupled to and / or arranged within the tubular section.

[0060] Preferably, the bioreactor includes at least one port configured for accessing the reactor volume, preferably wherein the at least one port is adapted for inserting a probe, such as a temperature probe, a pH probe, and / or an oxygen probe. The at least one port may be arranged at a side of the reactor body and / or at a bottom or top section of the reactor body.

[0061] Preferably, the at least one port is arranged above the tapering bottom section, and / or above an assumed maximal filling level of the reactor body.

[0062] Preferably, the bioreactor includes a sparger, preferably an air sparger, more preferably an oxygen sparger, for supplying air, preferably oxygen, to the reactor volume.

[0063] Preferably, the sparger includes one or more openings configured for producing bubbles, such as air and / or oxygen bubbles, having a size of less than 10 mm, preferably less than 5 mm, preferably less than 4 mm, more preferably less than 2 mm, more preferably of less than 1 mm, and / or more preferably in a range between about 0.1 mm and about 4 mm, even more preferably in a range between 0.1 mm and 1 mm. The inventors have found that the fine bubbles (such as microbubbles) generated by the sparger significantly enhance gas transfer efficiency, thereby promoting protein growth. For example, the fine bubbles may increase the dissolved oxygen in the liquid, preferably such that a dissolved oxygen saturation between 20% and 30%, or even higher, may be maintained.

[0064] However, the inventors have found that these fine bubbles alone may be insufficient for effective mixing or stirring within the reactor volume. For example, using a sparger with a pore size of 0.22 micrometers and / or a ring sparger with a pore size of 1 mm showed insufficient internal recirculation, particularly in fluids with high viscosity, such as during growth of mycelia.

[0065] Surprisingly, the inventors have discovered that combining the fine bubbles generated by the sparger with the larger bubbles produced by the exit openings of the first fluid channel— which enhance stirring and mixing— yields a synergistic effect. This integrated approach simultaneously optimizes gas transfer (using the fine bubbles) and mixing (using the large bubbles), thereby significantly improving overall reactor performance without the need of a separate stirring device.

[0066] Preferably, the sparger is arranged in the tapering bottom section of the reactor wall.

[0067] Alternatively or additionally, the sparger may be arranged within and / or through the cap, preferably within at least one of the first fluid channel and the second fluid channel, more preferably within the first fluid channel. As a result, it does not interfere with the internal environment of the reactor, thereby minimizing any potential negative impact on the length of the produced protein or mycelia. Furthermore, this arrangement has been observed to promote increased protein growth.

[0068] Preferably, the bioreactor comprises a top opening provided in the reactor wall and being configured for providing access to the reactor volume, and a lid for closing the top opening. Preferably, the lid includes a lid body with one or more channels extending therethrough, and one or more connection ports connected to each of the channels. Preferably, at least one channel extending through the lid body is a fluid channel for transferring fluid between the inside and the outside of the reactor body. The respective connection port may be fluidly connected to the channel. The at least one connection port may be configured for connecting to a respective fluid line for transferring fluid into or out of the reactor body. Preferably, a plurality of connection ports is provided. The connection ports may me fluidly connected to the same channel or to individual channels. Fluid lines such as water, media, inoculum, antifoam, base, acid etc. may be connected to the connection ports.

[0069] Preferably, at least one channel extending through the lid body and / or its respective connection port is configured for connecting to a probe. Probes such as temperature, pH, oxygen etc. may be connected to the one or more connection ports. At least one of the fluid channels may be configured for transferring fluid and extending a probe therethrough.

[0070] Preferably, the lid comprises a plurality of channels. Preferably at least one of the channels is a fluid channel and at least another one of the channels is configured to be provided with a probe.

[0071] Preferably, in lab scale bioreactors, probes may be coupled to the connection ports of the lid. Preferably, in industrial scale bioreactors, fluid lines may be coupled to the connection ports of the lid.

[0072] Preferably, the lid is screwed into the top opening, and / or the lid is plug-fitted and / or press-fitted into the top opening.

[0073] Preferably, the lid is sealingly fitted to (e.g., into or onto) the top opening.

[0074] Preferably, the lid is removably fitted to (e.g., into or into) the top opening.

[0075] Preferably, the reactor volume is in a range between 1 liter and 1000 hectoliters, and / or a reactor height is in a range between 0.225 m and 15 m or even higher, and / or the cap has a height in a range between 5 cm and 20 cm or even smaller. In other words, lab scale bioreactors as well as industrial scale bioreactors may be retrofitted with the cap. The cap itself may have a relatively small height between 5 cm and 20 cm such that retrofitting of the cap may be possible over a wide range of bioreactor types, sizes and installation spaces. Preferably, the reactor volume is 50 hectoliters or more, 100 hectoliters or more, or even 500 hectoliters or more. In other words, as explained below, the invention preferably allows usage and / or retrofitting of large bioreactors.

[0076] Preferably, the bioreactor is adapted for fermentation, preferably submerged fermentation, more preferably for submerged fermentation of microbial protein / fungal mycelia used as food analogue, food stuff or food alternative, such as meat alternative.

[0077] Preferably, the bottom opening is arranged centrally on the reactor body and / or at a lowest point of the bottom section. Alternatively, the bottom opening may be arranged at any position in the tapering bottom section. When the cap is installed at location where the inner surface of the reactor wall is curved, the cap may have a curved top face that is aligned with the curved inner surface of the reactor wall. The cap may be installed flush with the inner surface of the reactor wall. Preferably, no niches, steps etc. between the top face and the inner surface of the reactor wall are present after installation of the cap.

[0078] According to a second aspect of the present invention, a method of producing a protein, such as a mycoprotein and / or mycelium fibers, preferably mycelium, in a bioreactor is proposed. The bioreactor includes a reactor body with a reactor wall, the reactor wall confining a reactor volume for incubating an inoculum configured for producing the protein, such as mycelium, the reactor wall having a tapering bottom section; a bottom opening provided in the reactor wall at the tapering bottom section and being configured for providing access to the reactor volume; and a cap for closing the bottom opening. The cap includes a top face, and a first fluid channel extending through the cap, wherein the first fluid channel is configured for transferring fluid between an inside of the reactor body and an outside of the reactor body, and wherein an exit opening of the first fluid channel is arranged flush with the top face of the cap. The method comprises the steps of incubating the inoculum within the reactor body, and aerating the inoculum by providing air and / or oxygen through the first fluid channel of the cap. The first fluid channel may thus be an aeration channel but may be used for other purposes as well.

[0079] The method of the present invention is based at least partially on the idea that a bioreactor as explained in connection with the first aspect, could be used for producing mycoprotein, such as mycelium, by incubation an inoculum in the reactor volume and aerating the inoculum through the first fluid channel.

[0080] Preferably, the method includes the step of feeding cultivation medium into the reactor body, preferably wherein the cultivation medium is fed through a lid closing a top opening of the reactor body, and / or through a second fluid channel arranged in the cap, and aerating the mixture of inoculum and cultivation medium by providing air and / or oxygen through the first fluid channel of the cap. In other words, cultivation medium is fed into the reactor body and the mixture of inoculum and medium is aerated through the first fluid channel of the cap.

[0081] Preferably, the method includes the step of continuously aerating the mixture of inoculum and cultivation medium for enabling growth of proteins, preferably for enabling the growth of fungal cells or fungal mycelium, such as germination of fungal spores followed by extensive mycelium growth, within the bioreactor. In other words, a submerged fermentation process may be used for growing mycelium. The fermentation process may be used, for example, in a known brewery reactor which has been retrofitted by adding a flush aeration cap at the bottom opening of the reactor.

[0082] Preferably, aerating is such that air and / or oxygen bubbles are produced at the exit opening of the first fluid channel for stirring the mixture. Preferably, aerating includes providing air and / or oxygen bubbles of at least 10 mm, more preferably at least 20 mm, even more preferably at least 30 mm at the exit opening of the first fluid channel for stirring the mixture.

[0083] Preferably, aerating includes providing air and / or oxygen bubbles in a range between 10 mm and 40 mm, preferably between 10 mm and 30 mm, more preferably between 10 mm and 20 mm.

[0084] Preferably, aerating includes providing an air lift or bubble column within the reactor volume, preferably for creating a stir, and more preferably a recirculation zone, within the reactor volume.

[0085] A throughput of air / oxygen may be adapted by adjusting a geometry of the first fluid channel, and / or a number of exit openings of the first fluid channel, and / or a pressure and / or a flow rate of air / oxygen through the first fluid channel.

[0086] Preferably, a concentration of oxygen in the air used for aeration is increased when compared to an oxygen level in ambient air. This may allow to enhance cell growth. Preferably, a concentration of oxygen in the air used for aeration is at least 22%, preferably at least 23%, more preferably at least 25%.

[0087] Preferably, the method further comprises a step of providing additional aeration within the reactor volume using a sparger, the additional aeration being in addition to the aeration provided by the air and / or oxygen through the first fluid channel.

[0088] Preferably, wherein the step of providing additional aeration includes providing air and / or oxygen bubbles having a size of less than 10 mm, preferably less than 5 mm, more preferably in a range between about 1 mm and about 4 mm.

[0089] Preferably, wherein the additional aeration is provided at the tapered bottom section of the reactor wall and / or within (and / or through) the cap, preferably within at least one of the first and second fluid channel, more preferably within the first fluid channel.

[0090] Preferably, the bioreactor may be operated in fed-batch mode. Preferably, the amount of cultivation medium in the bioreactor in such fed-batch mode is increased over a predetermined amount of time, preferably wherein the amount of medium is increased over a time of at least 5 hours, more preferably at least 12 hours, even more preferably at least 24 hours. Preferably, the time is 48 hours or less, more preferably the time is 30 hours or less. In other words, the bioreactor may be operated such that continuous and sustained growth of mycoprotein is obtained. Operating the bioreactor in fed-batch mode may result in higher growth rates compared to a batch mode operation as the inoculum and / or cells are kept in a predetermined growth regime. In addition, substrate inhibition may be reduced as in the fed-batch mode, cultivation medium is fed in a ramp-up and not as one-off (as is the case in a batch-mode operation of the reactor).

[0091] The skilled reader will understand that a batch-mode operation of the bioreactor may work as well. A batchmode operation may be desirable, for example, for producing bioproducts such as enzymes or vitamins using filamentous fungi. A batch-mode may, however, not be the preferred operation mode when it comes to producing fungal mycelium as food or meat alternative.

[0092] Preferably, the cultivation medium is fed such that exponential growth is maintained during feeding of the medium.

[0093] Preferably, the method includes the step of harvesting produced proteins, such as produced mycoproteins, from the bioreactor, preferably wherein harvesting is done through a second fluid channel arranged in the cap, preferably wherein the second fluid channel has a diameter of at least 10 mm, more preferably at least 15 mm, to prevent clogging during harvesting. In other words, preferably produced (myco)proteins are harvested through the second fluid channel, which may be adapted to a high throughput without clogging.

[0094] Preferably, the method includes the steps of harvesting not more than a predetermined percentage of produced proteins, such as harvesting between 60% and 95%, preferably 80% and 95%, and, after harvesting, feeding further cultivation medium into the reactor body for producing further protein within the same bioreactor. In other words, the bioreactor may be operated in semi-continuous fed-batch mode. This operation mode improves efficiency and / or reduces downtime of the reactor as no emptying and / or cleaning of the bioreactor in-between different batches is necessary.

[0095] Preferably, the inoculum may be provided through a lid that is configured to close a top opening of the reactor body and / or through a second or third fluid channel that is provided in the cap closing the bottom opening of the bioreactor.

[0096] Preferably, the bioreactor may be operated in a continuous mode, the continuous mode comprising a step of harvesting a first portion of the produced protein, such as mycelium, while a second portion of the produced protein, such as mycelium, remains in the bioreactor, preferably in the reactor body, a step of feeding further cultivation medium into the reactor body, and a step of further incubating the second portion of the produced protein in the reactor body. Two or all three of these steps may be performed essentially simultaneously or alternatingly. Preferably, harvesting and feeding is done essentially simultaneously or alternatingly. More preferably harvesting and feeding is adapted such that cell growth is maintained, preferably at exponential phase or rate, during harvesting and feeding. In other words, incubation of the second portion may take place while feeding further cultivation medium into the reactor and / or during harvesting the first portion.

[0097] Preferably, the bioreactor is operated in the continuous mode, or in a semi-continuous mode, over a time period of 48 hours or more, preferably over a time period of 100 hours or more, more preferably over a time period of 500 hours or more, such as 800 hours or even longer.

[0098] Preferably, the method includes producing mycelium fibers, such as mycoprotein fibers and / or hyphae, at a production rate of at least 10 g / L / d, preferably at least 15 g / L / d, more preferably at least 20 g / L / d, even more preferably at least 30 g / L / d, preferably wherein the produced mycelium fibers (in particular, the mycelium hyphae) have an average length of at least 2 mm, preferably at least 3 mm, preferably at least 4 mm. .

[0099] As the skilled reader will appreciate, any preferred embodiment of the bioreactor according to the first aspect may be combined with any preferred embodiment of the method according to the second aspect.

[0100] Any preferred embodiment of the method according to the second aspect may be combined with any preferred embodiment of the bioreactor according to the first aspect.

[0101] A third aspect of the present invention is directed to a fungal biomass, preferably a mycoprotein biomass and / orfungal mycelium, such as mycelium fibers and / or mycelium hyphae, obtained by the method according to the second aspect or preferred embodiments thereof.

[0102] A fourth aspect of the present invention is directed to a fungal biomass, preferably a mycoprotein biomass and / or fungal mycelium, such as mycelium fibers and / or mycelium hyphae, obtained from a bioreactor according to the first aspect or preferred embodiments thereof, preferably operated using a method according to the second aspect or preferred embodiments thereof.

[0103] A fifth aspect of the present invention is directed to a fungal biomass, preferably a mycoprotein biomass and / or fungal mycelium, such as mycelium fibers and / or mycelium hyphae, wherein mycelium fibers, such as a mycoprotein fibers and / or mycelium hyphae, have at least one of: a minimum length of at least 0.4 mm, preferably at least 0.5 mm, more preferably at least 0.8 mm, more preferably at least 1 mm, more preferably at 1.5 mm, such as for example 1.97 mm; an average length of at least 2 mm, preferably at least 3 mm, preferably at least 4 mm; a maximum length of at least 5 mm, preferably at least 8 mm, more preferably at least 10 mm; an average length of 25 mm or less, preferably 20 mm or less, preferably 15 mm or less.

[0104] Brief description of the drawings

[0105] Figures 1A-B are schematic views of one example of a bioreactor according to the present invention.

[0106] Figure 2A-C are schematic views of another example of a bioreactor according to the present invention.

[0107] Figure 3 is a schematic view of bioreactor according to the present invention including a probe.

[0108] Figures 4A-C are schematic views of one example of a cap used in connection with the bioreactor.

[0109] Figures 5A-B are schematic views of another example of a cap.

[0110] Figure 6 is another schematic view of the cap of Figures 5A-B.

[0111] Figure 7 is a schematic view of another example of a bioreactor according to the present invention together with a detailed view of a lid used in connection with the bioreactor. Figures 8A-B are schematic views of another example of a cap used in connection with the bioreactor.

[0112] Figures 9 is a schematic view of a further example of a cap used in connection with a bioreactor according to the present invention.

[0113] Figures 10 is a schematic view of a further example of a bioreactor according to the present invention.

[0114] Figures 11 is a schematic view of a further example of a bioreactor according to the present invention.

[0115] Figure 12 is a schematic diagram of processing steps of an example of a method for producing protein according to the present invention.

[0116] Figure 13 is a schematic diagram of processing steps of another example of a method for producing protein according to the present invention.

[0117] Figure 14 is a schematic diagram of processing steps of another example of a method for producing protein according to the present invention.

[0118] Detailed description

[0119] Within the figures, same components are referenced by the same reference numerals.

[0120] Figures 1A-B show schematic views of a bioreactor 10. Figure 1A is a side view and Figure IB is a top view.

[0121] The bioreactor 10 may be used for producing any kind of protein. For example, the bioreactor 10 may be used for producing mycoproteins such a mycelium. Mycoproteins such as mycelium may be used for producing food analogues or food alternatives, such as mycelium-based meat, fish, dairy or the like.

[0122] The bioreactor 10 may be made from any suitable material, such as glass, stainless steel or other appropriate materials known in the food industry.

[0123] The bioreactor 10 includes a reactor body 12 having a reactor wall 14. The reactor wall 14 defines a reactor volume for incubating an inoculum. The reactor volume may be anything between 1 liter and several 1000 hectoliters, depending on the size and use of the bioreactor 10. The reactor height may be anything between 0.2 meters and 15 meters, depending on the use of the bioreactor 10 in lab scale or industrial scale applications.

[0124] The inoculum which is incubated inside the reactor body 12 may be understood as a starter culture for starting a fermentation process such as fermentation of fungal mycelia or other proteins. The inoculum may include spores, hypha or mycelium suitable for producing mycoproteins using submerged fermentation. Details on the fermentation process and / or the method of producing proteins and preferably mycoproteins using the bioreactor 10 will be explained in connection with Figures 9 to 11. The reactor wall 14 includes a tapering bottom section 16. The tapering bottom section 16 may be a conical bottom section, a concave bottom section, a converging bottom section or any other tapering bottom section. The bottom section 16 includes a bottom opening 18. The bottom opening 18 provides access to the reactor volume. In the specific embodiment shown, the bottom opening 18 is arranged centrally on the reactor body 12 and at the lowest position of the bottom section 16. In other embodiments this may not necessarily be the case.

[0125] The reactor body 12 shown in Figure 1A may have a similar shape than known brewery fermenters. Such fermenters usually have a cylindrical vessel body with a conically shaped lower section similar to the bottom section 16. Brewery fermenters may also have a central bottom opening similar to the bottom opening 18. In known brewery fermenters, yeast, hop, water etc. may be inserted into the reactor body 12 via the central bottom opening. After fermentation, fermented proteins such as mash or beer may be harvested through the central bottom opening, as known to a person skilled in the art.

[0126] In the bioreactor 10 according to the present invention, the bottom opening 18 is covered by a cap 20. The cap 20 is, e.g., screwed onto the bottom opening 20 and closes the bottom opening 18. The cap 20 may be in a screwed, clamped, bayonet etc. connection with the bottom opening 18 for closing the same, preferably sealingly closing the same. The cap 20 may be removably fitted to the bottom opening 18. In other words, the cap 20 may be removable from the reactor body 12 without destroying the reactor body 12. For ease of understanding, in Figure 1A, the cap 20 is shown removed from the bottom opening 18. During use of the bioreactor 10, the cap 20 will be fitted to the bottom opening 18 and cover the bottom opening 18 for closing the same.

[0127] The cap 20 closes the reactor volume while providing access to the reactor volume, for example, for aerating ingredients contained inside the reactor body 12. The cap 20 may include at least one fluid channel through which air and / or oxygen may be provided. The air and / or oxygen may be used for aerating the inoculum and / or a mixture of inoculum and cultivation medium present in the reactor volume. The aeration may be done such that air and / or oxygen bubbles may be produced, as indicated in Figure 1A. The air and / or oxygen bubbles may provide a swirl and / or circulation within the reactor volume for mixing the ingredients contained inside the reactor body 12. The cap 20 will be discussed in more detail further below.

[0128] The air and / or oxygen bubbles may be large enough to produce an air lift effect and / or a stirring effect within the reactor volume.

[0129] The air and / or oxygen bubbles may have a size (such as a diameter or comparable / similar dimension) of (at least on average of a produced bubble size distribution) at least 10 mm, preferably at least 20 mm, more preferably at least 30mm, , and / or in a range between 10 mm and 40 mm, preferably between 10 mm and 30 mm, more preferably between 10 mm and 20 mm. The cap 20 may include a fluid channel (such as a first fluid channel) and / or exit opening(s) having a diameter adapted to produce such larger air and / or oxygen bubbles for a given operating range of the bioreactor 10.

[0130] The bioreactor 10 may further include ports 22, 24 configured for further functions and / or needs, such as but not limited to feeding cultivation medium, inoculation, exhaust connection, connecting probes, lines, or others. Probes may be pH-probes, temperature probes, or others. Particularly in bench top bioreactors the probes may be located on or at the top. In large-scale fermenters, the ports 22, 24 may be located at any position in contact with the reactor volume.

[0131] Figure IB shows a schematic top view of the bioreactor 10. As can be seen, ports 22 may protrude from the side and ports 24 may protrude from on top.

[0132] The skilled reader will understand that ports 22, 24 may be used for any purpose and / or may be arranged at any suitable location on the reactor body 12. Thus, the number of ports 22, 24 and / or the locations of ports 22, 24 may be different in other embodiments. It may be possible that, in other embodiments, the bioreactor 10 may not include ports 22, 24. Ports 22, 24 may thus be optional to the bioreactor 10.

[0133] Figures 2A-C show schematic views of another bioreactor 10. Figure 2A is a side view, Figure 2B is a top view, and Figure 2C is a detailed view of the bioreactor 10. The bioreactor 10 show in Figures 2A-C may be a small- scale bioreactor, but not necessarily.

[0134] The bioreactor 10 of Figures 2A-C includes the reactor body 12 with the reactor wall 14, the tapering bottom section 16 and ports 22. Cap 20 is used to cover the bottom opening 18. As with the previous embodiment, cap 20 may be used for aeration, as indicated by the air bubbles. Compared to the previous embodiment shown in connection with Figures 1A-B, the embodiment of Figures 2A-C includes a lid 26. The lid 26 is used for closing a top opening of the reactor wall 14. The lid 26 may be fitted to the top opening in any suitable way, such as but not limited to screwing, plug-fitting, and press-fitting. The lid 26 may be sealingly fitted to the top opening and / or removably fitted to the top opening.

[0135] Figure 2B shows a top view of the bioreactor 10 with the ports 22 and lid 26.

[0136] Figure 2C shows a detailed view of the lid 26. The lid 26 may include one or more channels for transferring fluid between an inside and an outside of the reactor body 12. In the specific embodiment shown, multiple ports 28 may be included in the lid 26. These ports 28 are fluidly connected to the channels and may be used for connection to a fluid line and / or a probe used in connection with the bioreactor 10. In the specific embodiment shown, the ports 28 are arranged in a certain pattern and / or at certain locations on the lid 26. For example, port 28a may be used to provide a base to the reactor volume, port 28b may be used for stirring, port 28c may be used to provide acid to the reactor volume, port 28d may be used to connect a probe such as oxygen or pH-probes, port 28e may be used to feed cultivation medium to the reactor volume, port 28f may be used to provide antifoam to the reactor volume, and port 28g may be used to connect to another probe.

[0137] The skilled reader will understand that in other embodiments not shown, the number and / or arrangement of ports 28 on the lid 26 may be different. The arrangement of ports 28 shown in Figure 2C may thus not be interpreted as limiting the scope of this disclosure.

[0138] Figure 3 shows another schematic view of the bioreactor 10 of Figures 2A-C. As can be seen in Figure 3, a probe 30 is inserted through the lid 26. The probe 30 may be coupled to a port of the lid 26 using an appropriate adapter 32.

[0139] Referring to Figures 4A-C, schematic views of an example of cap 20 are shown.

[0140] Cap 20 includes a main body 34 configured to be coupled to the bottom opening of the bioreactor 10 for covering and closing the same. The cap 20 further includes a first fluid channel 36 and in the specific embodiment shown another second fluid channel 38. The first fluid channel 36 may be used for aeration by providing air and / or oxygen through the first fluid channel 36. The second fluid channel 38 may be used for providing cultivation medium to the reactor volume and / or for harvesting produced proteins. Additional fluid channels not shown in Figures 4A-C may be provided in the cap 20. Valves (not shown), control units (not shown), and / or other devices may be coupled to the cap 20 for opening and / or closing the fluid channels provided in the cap 20, and / or for routing fluids between fluid channels.

[0141] In the specific embodiment shown, the first fluid channel 36 includes a first fluid channel axis 40 which is arranged at an angled orientation with respect to a main axis 42. Main axis 42 may be the main axis of the reactor body and / or the tapering bottom section, and / or may be the main axis of the second fluid channel 38. The second fluid channel 38 may be arranged such that its main axis is parallel to and / or coincides with main axis 42. However, other arrangements are also possible.

[0142] An angle 44 between the main axis 42 and the first fluid channel axis 40 may be in a range between 10° to 60°, preferably between 20° to 30°. The angle may depend on the installation space that is provided underneath the reactor body.

[0143] The first fluid channel 36 further includes an exit opening 46 and the second fluid channel 38 includes another exit opening 48. As can be seen in Figures 4B-C, the exit opening 46 of the first fluid channel 36 is arranged flush with a top face 50 of the main body 34, and the exit opening 48 of the second fluid channel 38 is arranged flush with the top face 50 of the main body 34. Preferably, no niches or steps are provided on the top face 50. The top face 50 is smooth and in the specific embodiment shown also planar. The cap 20 is adapted such that the top face 50 smoothly transitions into an inner surface of the reactor wall without having dents, niches, steps in-between. During production of mycoproteins, fungal mycelia may adhere to niches, steps etc. and may clog the reactor. A smooth and flush inner surface of the reactor body reduces the likelihood of fungal mycelium adhering to these areas so that clogging of the reactor in these regions may be prevented or at least limited.

[0144] The exit opening 46 and / or the first fluid channel 36 is dimensioned such that air and / or oxygen bubbles of at least 10 mm, preferably at least 20 mm, more preferably at least 30 mm, and / or in a range between 10 mm and 40 mm, preferably between 10 mm and 30 mm, more preferably between 10 mm and 20 mm, at least on average, are provided at the exit opening 46 of the first fluid channel 36. The exit opening 46 and / or first channel 36 is dimensioned to provide air and / or oxygen bubbles large enough for providing a stir and / or air bubble column or air lift within the reactor volume. In other words, the air and / or oxygen bubbles exiting the exit opening 46 may provide a stir and / or a recirculation within the reactor volume. In this case, the reactor volume may be devoid of an additional stirrer (such as devoid of a mechanical stirrer, e.g. a mechanical stirrer that rotates around the shaft). In this manner, average fiber lengths of 4.15 mm and more were obtainable. This contrasts with stirred bioreactors of otherwise equivalent design, where the average length was found to be around 1.77 mm.

[0145] A diameter of the first fluid channel 36 and / or the exit opening 46 may be in a range between 25 mm and 60 mm, preferably in a range between 25 mm and 50 mm.

[0146] As can be seen in Figures 4A-C, a diameter of the second fluid channel 38 is larger than a diameter of the first fluid channel 36. For example, the diameter of the second fluid channel 38 may be larger by a factor of 4, 6, 8, 10 or more than the diameter of the first fluid channel 36. The diameter of the second fluid channel 38 may be at least 10 mm, preferably at least 15 mm such as a DN 15-20 or larger, such as DN32, or even larger. There may be no fixed upper end of a diameter range for the second fluid channel 38. The diameter of the second fluid channel 38 may be adapted such that produced mycelia may be harvested through the second fluid channel 38 without clogging of the second fluid channel 38. Typically, mycoprotein-broths such as mycelia-broths are rather viscous and / or thick. In addition, strands of mycelia may get tangled in small diameter discharge lines. By having a diameter of at least 10 mm, preferably at least 15 mm or larger, clogging of the second fluid channel 38 during harvesting of mycelia-broths may be prevented.

[0147] The second fluid channel 38 may include an outer thread 52 for coupling to a discharge line and / or a valve section coupled to a plurality of lines. The valve section may also include a DN 15-20, or larger, such as DN32, or even larger.

[0148] A height 54 of the cap 20 may be, for example, in a range between 5 cm and 20 cm which ensures that the cap 20 may be retrofitted underneath the reactor body in a large range of installation spaces. The skilled reader will understand that the cap 20 may have any suitable height that is adapted to the actual installation space available.

[0149] Figures 5A-B show schematic views of another example of cap 20. In the embodiment of Figures 5A-B, the first fluid channel 36 includes a plurality of exit openings 46 (only one being shown in the cross section of Fig. 5B). As can be seen in Figure 5B, each exit opening 46 is arranged flush with the top face 50 of the cap 20. The first fluid channel 36 includes a main channel portion 56 and a ring channel portion 58. The ring channel portion 58 fluidly connections the plurality of exit openings 46 with the main channel portion 56. As can be further seen in Figure 5B, the exit openings 46 extend at exit openings axes 60 that may be oblique to the main axis 42. An angle 62 between the exit opening axes 60 and the main axis 42 may be in a range between 10° to 60°, preferably between 20° to 30°.

[0150] An end portion of the section(s) of the first fluid channel 36 extending proximate the exit opening(s) 46 may extend in a direction having a directional component around the circumference of the cap 20 and / or the top surface 50. In other words, the exit opening(s) 46 may be configured to eject air bubbles in a direction that is tangential to the inner surface of the bottom section 16 and / or the reactor body 12. This is schematically shown by dashed arrows in Figure 6. Figure 6 shows a schematic top view of the cap 20 of Figures 5A-B.

[0151] The exit openings 46 may be dimensioned such that air and / or oxygen bubbles of at least 10 mm, preferably at least 20 mm, more preferably at least 30 mm, and / or in a range between 10 mm and 40 mm, preferably between 10 mm and 30 mm, more preferably between 10 mm and 20 mm, at least on average, are provided at the exit openings 46. The exit openings 46 may each be dimensioned to provide air and / or oxygen bubbles large enough for providing a stir and / or air bubble column or air lift within the reactor volume. In other words, the air and / or oxygen bubbles exiting the exit openings 46 may provide a stir and / or a recirculation within the reactor volume so that an additional stirrer or stirring device within the reactor volume may be superfluous.

[0152] A diameter of the exit openings 46 may be in a range between 25 mm and 60 mm, preferably in range between 25 mm and 50 mm.

[0153] Alternatively or additionally, at least some of the exit openings 46 may function as a sparger and may be dimensioned to produce bubbles of less than 4 mm in size (e.g., diameter) or less than 2 mm in size. For example, the diameter of the exit openings 46 functioning as a sparger may be smaller than 2 mm, preferably smaller than 1 mm.

[0154] One or more of the exit openings 46 may be dimensioned to create large bubbles (such as bubbles having an average diameter of at least 5 mm, preferably at least 8 mm, more preferably at least 10 mm, more preferably at least 20 mm, even more preferably at least 30 mm) while another one or more of the exit openings 46 may be dimensioned to create small bubbles (such as bubbles having an average diameter of less than 2 mm, preferably less than 1 mm).

[0155] The end portion of the section(s) of the first fluid channel 36 and / or the exit opening(s) 46 may be adjusted to generate a swirl within the reactor volume. The swirl may enhance mixing of the ingredients as explained above. The second fluid channel 38 may align with the main axis 42.

[0156] A height 54 of the cap 20 may be in a range between 5 cm and 20 cm.

[0157] As can be further seen in Figure 6, the exit openings 46 may be arranged in a predetermined pattern on the top face 50 of the cap 20. In the specific embodiment shown, the exit openings 46 are arranged in a circular pattern and / or concentric around the exit opening 48 of the second fluid channel 38. In other embodiments, this may not be the case.

[0158] As can be also seen in Figure 6, a diameter 64 of the exit opening 48 is considerably larger than a diameter of the exit openings 46, for example by a factor of 4, 6, 8, 10 or more. The diameter 64 of exit opening 48 and / or second fluid channel 38 may be smaller than a diameter 66 of the main body 34 of the cap 20, such as by a factor of 2, 3, 4 or more.

[0159] Referring to Figure 7, a schematic view of another example of the bioreactor 10 is shown. The bioreactor 10 of Figure 7 includes the reactor body 12 with the reactor wall 14 and the tapering bottom section 16. The cap 20 is used for closing the bottom opening 18. Ports such as ports 22 may be provided at the side of the reactor body 12. A top opening of the reactor wall 14 may be closed by a lid 68. The lid 68 may include multiple ports 70 which may be configured for coupling to lines. A lid body may include a channel 72 which can be used for transferring fluid into and out of the reactor volume. The multiple ports 70 may be fluidly connected to the channel 72. Ports 70 may be used for adding water, cultivation medium, inoculum, antifoam, base, acid or other fluids. Ports 70 may also be used for connection of probes, if suitable. The lid 68 may be connected to the reactor body 12 using a dairy coupling (e.g., in accordance with DIN 11851) or any other suitable coupling.

[0160] Referring to Figures 8A-B, schematic views of another example of a 20 cap are shown.

[0161] In the embodiment of Figures 8A-B, the cap 20 includes the first fluid channel 36 and the second fluid channel 38. The first fluid channel axis 40 is arranged at an angle 44 with the main axis 42. The cap 20 may be coupled to the reactor body using a dairy coupling (e.g., in accordance with DIN 11851) or any other suitable coupling.

[0162] The following are examples of methods for producing protein such as mycoprotein in a reactor such as the bioreactor 10. For ease of understanding, the method is explained with reference to the components of the bioreactor 10.

[0163] The cap 20 may be attached to the bioreactor via a threading or bayonet connection (not shown). Alternatively or additionally the cap 20 may be clamped to the bioreactor, e.g. via a coupling ring (not shown) that holds the cap 20 against the bioreactor.

[0164] Figure 9 shows another example of cap 20. In the example of Figure 9, the cap 20 may be an off-the-shelf component. In the example, shown, the component includes a female connection port 79 and a male connection port 80, as well as a tubular section 81 (Fig. 9 at the bottom schematically illustrates a male outer threading). In the example shown, the female connection port 79 is configured to be connected to the reactor wall 14 (see, e.g., Figure 1A) for coupling the cap 20 to the bottom opening 18. In the embodiment shown, the first fluid channel 36, which is preferably used for aeration, is coupled to the tubular section 81. The idea is to use standard off-the-shelf components and adapt those components to include an aeration channel (such as the first fluid channel 36).

[0165] In other embodiments, the component 79 may be a male connection port and component 80 may be a female connection port. In yet other embodiments, both components 79 or 80 may be female or male connection ports.

[0166] Figure 10 shows another example of a bioreactor 10. In the example of Figure 10, the bioreactor 10 includes a sparger 82. The sparger 82 can be used to provide additional aeration in addition to the aeration provided by the exit openings 46 of the cap 20 (see, e.g., Figures 4B, 6 or 8A).

[0167] The sparger 82 includes one or more openings, such as pores, configured for producing bubbles, such as air and / or oxygen bubbles in addition to any bubbles produced at the exit openings 46 of the cap 20.

[0168] Preferably, the bubbles produced by the sparger 82 have a size smaller than those bubbles provided at the exit opening or exit openings 46 of the cap 20.

[0169] Preferably, the bubbles produced by the sparger 82 have a size of less than 10 mm, preferably less than 5 mm, more preferably in a range between about 0.1 mm and about 4 mm, even more preferably in a range between 0.1 mm and 4 mm. It was found that the fine bubbles generated by the sparger 82 significantly enhance gas transfer efficiency, thereby promoting protein growth. However, these fine bubbles alone may be insufficient for effective mixing or stirring within the reactor volume. Surprisingly, when used in combination with the larger bubbles produced by the exit opening(s) 46 of the cap 20 —which facilitate stirring and mixing (see the description in relation to, e.g., Figures 4 and 6) —the fine bubbles of the sparger 82 provide a synergistic benefit. This combined approach optimizes both gas transfer and mixing, resulting in improved overall reactor performance.

[0170] In the example shown in Figure 10, the sparger 82 is arranged in the tapering bottom section 16 of the reactor wall 14.

[0171] In other examples not shown, the sparger 82 may be arranged in a different section of the reactor volume, e.g., closer to ports 22 and / or lid 26.

[0172] Figure 11 shows another example of a bioreactor 10. In the example of Figure 11, the bioreactor 10 includes sparger 82. In the example of Figure 11, the sparger 82 is arranged within the cap 20. In the example shown, the sparger 82 does not protrude into the reactor volume. This is beneficial as the sparger 82 does not interfere with the mixture, such as the protein, within the reactor volume. Hence, any potential impact on a length of the produced protein such as mycelia can be minimized. In the embodiment shown, the sparger 82 is arranged within the first fluid channel 36. In other embodiments not shown, the sparger 82 may be provided in the second fluid channel 38 (in case the second fluid channel 38 is also used for aeration).

[0173] The sparger 82 used in the embodiment of Figure 11 provides the smaller-sized bubbles to enhance protein growth as described in relation to Figure 10.

[0174] In the example of Figure 11, where the sparger 82 is arranged within the first fluid channel 36, the exit opening 46 may produce large air and / or oxygen bubbles for stirring as well as smaller-sized air and / or oxygen bubbles provided by the sparger 82 for dissolving oxygen. The sparger 82 may be provided as a sintered porous material within the first fluid channel 36.

[0175] In such case, the average size of larger air bubbles resulting from air that passes through the first fluid channel 36 and not through the sparger 82 (or, if applicable, through the second fluid channel 38 and not through the sparger 82) may have an average size (e.g., an average diameter) of 5 mm or more, 8 mm or more, or 10 mm or more.

[0176] The air bubbles resulting from air passing through the sparger 82 are preferably smaller than the air bubbles resulting from said air that does not pass through the sparger 82. The air bubbles resulting from air passing through the sparger 82 preferably have a size (e.g., an average diameter) of less than 5 mm, preferably less than 4 mm, preferably less than 2 mm or less than 1 mm.

[0177] While the sparger is shown to be mostly or even fully located within the first fluid channel 36 in Fig. 11, the sparger 82 does not necessarily have to be entirely within said first fluid channel 36 or the second fluid channel 38. For example, the sparger 82 may protrude from the first or second fluid channel 36, 38. While this may entail the risk of some biomass become stuck at the sparger, the bubbles coming out of the sparger and or the first and / or second fluid channel 36, 38 may minimize this effect, especially if the sparger does not extend far into the reactor volume.

[0178] Figure 12 shows a first example of a method for producing protein such as mycoprotein in the bioreactor 10.

[0179] In a first step 74, the inoculum is incubated within the reactor body 12. The inoculum may be introduced to the reactor volume via the bottom opening 18 and / or via the top opening of the reactor body. For example, the inoculum may be introduced via the second fluid channel 38 of the cap 20 and / or via any channels / ports of lids 26, 68. Additionally or alternatively, the inoculum may be provided through one of the ports 22, 24.

[0180] In a subsequent step 76, the inoculum is aerated by providing air and / or oxygen through the first fluid channel 40 of the cap 20. Retrofitting the cap 20 to known brewery fermenters may be sufficient for performing this basic method.

[0181] Figure 13 shows a more advanced method of producing protein such as mycoprotein in the bioreactor 10. In a first step 74, the inoculum is incubated within the reactor body 12. The inoculum may be provided as explained in connection with Figure 12.

[0182] In a second step 75, cultivation medium is fed into the reactor body 12. Cultivation medium may include solid, dissolved and / or liquid substrates containing, for example, a carbon source, a nitrogen source, mineral salts, trace materials and other suitable substrates, such as liquid brewery side streams. The cultivation medium may be provided as a one-off, for example, as a batch. Alternatively, the bioreactor 10 may be operated in fed-batch mode where an amount of cultivation medium may be increased over a predetermined amount of time. For example, the cultivation medium may be provided over a time of at least 5 hours, preferably at least 12 hours, preferably at least 24 hours. Preferably, the time may be 48 hours or less, preferably 30 hours or less. The amount of cultivation medium fed to the reactor volume may be such that sustained cell growth is maintained inside the reactor body 12. Preferably, cultivation medium may be fed such that an exponential cell growth regime is maintained.

[0183] The cultivation medium may be provided, for example, via a third fluid channel provided in the cap 20, via any of the ports 22, 24 of the reactor body 12. The cultivation medium may be fed through the second fluid channel 38 or through another, such as third, fluid channel provided in the cap 20. Additionally or alternatively, cultivation medium may be provided via any of the ports arranged in the lid 26, 68. In large- scale bioreactors 10, the cultivation medium and / or the inoculum may be provided at different ports and / or locations than in small-scale or lab-scale bioreactors 10.

[0184] The cap 20 may be provided with and / or may be coupled to appropriate valves / controls for operating the bioreactor 10 in batch and / or fed-batch mode. Known brewery fermenters may be retrofitted with the cap 20 and may be operated in batch and / or fed-batch mode using appropriate protocols so that known brewery fermenters may be used for producing proteins such as mycoproteins.

[0185] In a third step 76, a mixture of inoculum and cultivation medium is aerated by providing air and / or oxygen through the first fluid channel 40 of the cap 20. A throughput of air / oxygen may be adjusted according to the size or scale of broth that is fermented. Pressure and / or flow rate may be adjusted using any appropriate means. Aeration may be performed continuously. Aeration may be performed prior, after or simultaneously with the step of feeding cultivation medium.

[0186] Aeration may be such that air / oxygen bubbles may be created at the exit opening(s) 46 of the first fluid channel 36. The bubbles may introduce a swirl within the reactor volume. The swirl may enhance mixing and / or stirring of the ingredients provided in the reactor body 12. The swirl may ensure that the proteinbroth is stirred / mixed properly for limiting clogging within the bioreactor 10.

[0187] The exit opening(s) 46 may extend at oblique exit opening axes 40, 46 for guiding the air / oxygen into the reactor body 12 at obliques angles for introducing and / or enhancing a swirl within the reactor volume. A concentration of oxygen in the air provided through the first fluid channel 36 may be adapted to enhance cell growth. For example, the concentration of oxygen may be increased compared to an oxygen level provided in ambient air. The oxygen level in ambient air (at normal altitude) may be, for example, 21% and the concentration of oxygen in the air provided through the first fluid channel 36 may be, for example, increased by 10%, 20% or more, and / or may be at least 22%, preferably at least 23%, more preferably at least 24%.

[0188] In a fourth step 77, produced protein such as mycoprotein or mycelium broth is harvested from the bioreactor 10. Harvesting may be done via the second fluid channel 38. The diameter 64 of the second fluid channel 38 may be adjusted such that no clogging occurs during harvesting of the mycoprotein-broth. Usually, when mycelia is fermented, the broth is rather viscous and / or thick. The diameter 64 of the second fluid channel 38 may therefore be at least 10 mm, at least 15 mm or larger, as explained. Figure 14 shows processing steps of a further example of operating the bioreactor 10 for producing protein such as mycoprotein.

[0189] The processing steps 74-76 are the same as the processing steps explained in connection with Figure 13.

[0190] In the example of Figure 14, the bioreactor 10 is, however, operated in semi-continuous mode. In semi- continuous mode, a portion of produced mycoprotein is harvested. For example, between 60% and 95%, preferably between 80% and 95%, of produced mycoprotein is harvested in step 77. The remaining portion of produced mycoprotein remains inside the reactor body 12. The remaining portion may be used as another inoculum. In a further step 78, further cultivation medium may be fed into the reactor body for producing further protein. Step 78 may be performed after harvesting the predetermined amount of produced mycoprotein in step 77.

[0191] Alternatively or additionally, the bioreactor 10 may be operated in continuous mode.

[0192] As an example, in step 77 a first portion of the produced mycoprotein, such as mycelium, will be harvested while a second portion of the produced mycoprotein, such as mycelium, remains in the reactor body 12 and in step 78 further cultivation medium is fed into the reactor body and the second portion of the produced mycoprotein, such as mycelium, is further incubated in the reactor body 12. Preferably, steps 77 and 78 are done essentially simultaneously or alternatingly. More preferably steps 77 and 78 are adapted such that sustained cell growth is maintained during harvesting and feeding. Preferably, cell growth is maintained in the exponential regime. Harvesting and feeding steps 77, 78 may be performed over a time period of 48 hours or more, preferably over a time period of 100 hours or more, preferably over a time period of 500 hours or more, such as 800 hours or even longer.

[0193] The method steps explained in connection with Figures 12-14 may be performed with known brewery fermenters, for example, by retrofitting the known brewery fermenters with the cap 20 and / or the lid 26, 68. Retrofitted brewery ferments may be used to produce proteins, such as mycoproteins, in a batch, fed-batch, semi-continuous or continuous mode. The cap 20 has a small height in a range of about 5 cm to 20 cm. As a result, the cap 20 may be retrofitted underneath known brewery fermenters over a wide range of installation spaces. Lab-scale, small-scale and large-scale fermenters may be retrofitted and may be operated to produce mycoproteins such as mycelium in a low-cost and scalable way, for example, as building material and / or main ingredient for mycoprotein-based food analogues such as meat, fish, dairy, seafood, etc.

[0194] The methods explained may be followed by an optional processing step in which the mycoprotein biomass and / or fungal mycelium is separated from the broth.

[0195] The mycoprotein biomass and / or fungal mycelium may be deactivated in the aqueous phase by heat (e.g. 60- 75°C) before the separation step. The resulting mycoprotein biomass may then be used to create a food analogue or food alternative, such as mycelium-based meat, fish, dairy or the like.

[0196] The broth might be rich in proteins. It may contain between 40-60% protein content or even more. Proteins from the effluent broth can be further recovered for use in the food industry or as a food additive.

[0197] The skilled reader will understand that any of the examples shown in connection with Figures 1 to 11 may be combined with any examples shown in connection with Figures 12 to 14, and vice versa.

[0198] The following may be preferred aspects of this disclosure:

[0199] 1. A bioreactor (10) for producing a protein, such as a mycoprotein and / or mycelium fibers, the bioreactor (10) comprising: a reactor body (12) having a reactor wall (14), the reactor wall (14) confining a reactor volume for incubating an inoculum for producing the protein, the reactor wall (14) having a tapering bottom section (16), a bottom opening (18) provided in the reactor wall (14) at the tapering bottom section (16), and a cap (20) for covering the bottom opening (18), the cap (20) including a top face (50), and a first fluid channel (36) extending through the cap (20), wherein the first fluid channel (36) is configured for transferring fluid between an inside of the reactor body (12) and an outside of the reactor body (12), and wherein the first fluid channel (36) has an exit opening (46), preferably wherein the exit opening (46) of the first fluid channel (36) is arranged flush with the top face (50) of the cap (20).

[0200] 2. The bioreactor (10) of aspect 1, wherein the top face (50) is planar, and / or is configured for providing a smooth transition to an inner surface of the reactor wall (14).

[0201] 3. The bioreactor (10) of aspects 1 or 2, wherein, in a region around the bottom opening (18), the top face (50) of the cap (20) and an inner surface of the reactor wall (14) are arranged flush to one another.

[0202] 4. The bioreactor (10) of aspects 1, 2 or 3, wherein the reactor body (12) comprises a cylindrical body section, wherein the cylindrical body section and / or the tapering bottom section (16) have a main axis (42), the first fluid channel (36) has a first fluid channel axis (40), and the first fluid channel axis (40) has an angled orientation to the main axis (42), preferably wherein an angle (44) between the main axis (42) and the first fluid channel axis (40) is in a range between 10° to 60°, more preferably between 20° to 30°, and / or an angle (44) between the main axis (42) and the first fluid channel axis (40) is adapted for creating a fluid swirl within the reactor volume. The bioreactor (10) of any of aspects 1-4, wherein the exit opening (46) is configured for producing air and / or oxygen bubbles having a size of at least 5 mm, preferably at least 8 mm, more preferably at least 10 mm, more preferably at least 20 mm, even more preferably at least 30 mm. The bioreactor (10) of any of aspects 1-5, wherein the exit opening (46) is delimited by an inner wall of the first fluid channel (36), preferably wherein the exit opening (46) has a diameter of at least 10 mm, preferably at least 15 mm, more preferably at least 20 mm, more preferably at least 25 mm. The bioreactor (10) of any one of the previous aspects, wherein the first fluid channel (36) includes a plurality of exit openings (46), each exit opening (46) being arranged flush with the top face (50) of the cap (20). The bioreactor (10) of any one of aspects 5 to 7, wherein the first fluid channel (36) includes a main channel portion (56) and a ring channel portion (58), and the ring channel portion (58) fluidly connects the main channel portion (56) with the plurality of exit openings (46). The bioreactor (10) of any one of aspects 5 to 8, wherein the exit openings (46) are arranged in a predetermined pattern on the top face (50) of the cap (20), preferably wherein the predetermined pattern is a circular pattern around an imaginary central point; and / or wherein the exit openings (46) extend at exit opening axes (60) that are oblique to the main axis (42), preferably wherein the exit opening axes (60) are arranged at an angle (62) to the main axis (42), the angle (62) being in a range between 10° to 60°, more preferably between 20° to 30°, more preferably wherein the angle (62) is configured to create a fluid swirl within the reactor volume. The bioreactor (10) of any one of the preceding aspects, wherein the reactor volume is free of a stirring device, preferably free of a movable stirring device such as an impeller. The bioreactor (10) of any one of the preceding aspects, wherein the cap (20) includes a second fluid channel (38) that is separate from the first fluid channel (36), the second fluid channel (38) extending through the cap (20) and being configured for transferring fluid between the inside and the outside of the reactor body (12), and wherein an exit opening (48) of the second fluid channel (38) is arranged flush with the top face (50) of the cap (20). The bioreactor (10) of aspect 11, wherein the second fluid channel (38) is configured for discharging proteins and / or a broth including proteins, such as mycoproteins, from the reactor body (12), and / or the second fluid channel (38) aligns in parallel with a central or main axis (42) of the reactor body (12), and / or the first fluid channel (36) is configured for aerating a mixture of inoculum and a cultivation medium in the reactor volume, and / or the second fluid channel (38) has a diameter (64) of at least 10 mm, more preferably at least 15 mm, and / or the second fluid channel (38) has a diameter (64) of 15 to 20 mm or larger such as 32 mm or even larger. The bioreactor (10) of aspects 11 or 12, wherein a diameter (64) of the second fluid channel (38) is larger than a diameter of the first fluid channel (36) and / or an exit opening (46) of the first fluid channel (36), and / or a diameter (64) of the second fluid channel (38) is by a factor of 4, 6, 8, 10 or more, larger than a diameter of the first fluid channel (36) and / or an exit opening (46) of the first fluid channel (36). The bioreactor (10) of any one of aspects 11-13 in combination with any one of aspects 6-10, wherein the exit openings (46) of the first fluid channel (36) are arranged in a predetermined pattern around the exit opening (48) of the second fluid channel (38), and / or the exit openings (46) of the first fluid channel (36) are arranged in a circular pattern and / or in a concentric manner around the exit opening (48) of the second fluid channel (38). The bioreactor (38) of any one of the preceding aspects, wherein the cap (20) is screwed, or clamped onto and / or into the bottom opening (18), and / or the cap (20) and the bottom opening (18) are connected to each other via a threading, a bayonet connection, or a clamping mechanism, and / or the cap (20) includes an outer thread adapted to an inner thread of the reactor wall (14) at the bottom opening (18), and / or the cap (20) is sealingly fitted into the bottom opening (18), and / or the cap (20) is removably fitted, preferably threadedly fitted, into the bottom opening (18), and / or the cap (20) includes a port or a coupling for coupling at least one pipe or line, preferably a plurality of pipes or lines, to the cap (20), preferably wherein the cap (20) is configured for coupling to at least one pipe or line, preferably using a milk-coupling. The bioreactor (10) of any one of the preceding aspects, wherein the bioreactor (10) includes at least one port (22, 24) configured for accessing the reactor volume, preferably wherein the at least one 1 port (22, 28) is adapted for inserting a probe, such as a temperature probe, a pH probe, and / or an oxygen probe. The bioreactor (10) of aspect 16, wherein the at least one port (22, 24) is arranged above the tapering bottom section (16), and / or above an assumed maximal filling level of the reactor body (12). The bioreactor (10) of any one of the preceding aspects, further comprising: a top opening provided in the reactor wall (14) and being configured for providing access to the reactor volume, and a lid (26, 68) for closing the top opening, the lid (26, 68) including a lid body with a channel (72) for transferring fluid between the inside and the outside of the reactor body (12), and at least one connection port (28, 70) fluidly connected to the channel, wherein the at least one connection port (28, 70) is configured for connecting to a respective fluid line for transferring fluid into or out of the reactor body, and / or is configured for connecting to a probe (30). The bioreactor (10) of aspect 18, wherein the lid (26, 68) is screwed into the top opening, and / or the lid (26, 68) is plug-fitted and / or press-fitted into the top opening, and / or the lid (26, 68) is sealingly fitted into the lid opening, and / or the lid (26, 68) is removably fitted into the top opening. The bioreactor (10) of any one of the preceding aspects, wherein the reactor volume is in a range between 1 liter and 1000 hectoliters, and / or a reactor height is in a range between 0.225 m and 15 m, or even larger, and / or the cap (20) has a height (54) in a range between 5 cm and 20 cm. The bioreactor (10) of any one of the preceding aspects, wherein the bioreactor (10) is adapted for fermentation, preferably submerged fermentation, more preferably for submerged fermentation of microbial protein / fungal mycelia used as food analogue, food stuff or food alternative, such as mycoprotein-based food analogues, such as meat, fish, dairy, seafood etc. The bioreactor (10) of any one of the preceding aspects, wherein the bottom opening (18) is arranged at a lowest point of the bottom section (16). The bioreactor (10) of any one of the preceding aspects, wherein the bottom opening (18) is arranged centrally on the reactor body (12). The bioreactor (10) of any one of the preceding aspects, further comprising a sparger (82), preferably an air sparger, more preferably an oxygen sparger, for supplying air, preferably oxygen, to the reactor volume. The bioreactor (10) of aspect 24, wherein the sparger (82) includes one or more openings configured for producing bubbles, such as air and / or oxygen bubbles, having a size of less than 10 mm, preferably less than 5 mm, more preferably less than 2 mm, more preferably less than 1 mm; and / or in a range between about 0.1 mm and about 4 mm, such as in a range between about 0.1 mm and about 1 mm. The bioreactor (10) of aspect 24 or aspect 25, wherein the sparger (82) is arranged in the tapering bottom (16) section of the reactor wall (14). The bioreactor (10) of aspect 24 or aspect 25, wherein the sparger (82) is arranged within the cap (20). The bioreactor (10) of aspect 26, preferably in combination with any one of aspect 12-14, wherein the sparger (82) is arranged within at least one of the first fluid channel (36) and the second fluid channel (38), preferably within the first fluid channel (36). The bioreactor (10) of any one of the preceding aspects, wherein the bioreactor (10) is configured for producing mycelium fibers, such as mycoprotein fibers and / or hyphae, having an average length of at least 2 mm, preferably at least 3 mm, more preferably at least 4 mm, even more preferably at least

[0203] 5 mm, or even more than that. The bioreactor (10) of any one of the preceding aspects, wherein the bioreactor (10) is configured for producing mycelium fibers, such as mycoprotein fibers and / or hyphae, with a production rate of at least 10 g / L / d, preferably at least 15 g / L / d, more preferably at least 20 g / L / d, even more preferably at least 30 g / L / d, preferably wherein mycelium fibers, such as mycoprotein fibers and / or hyphae produced with such production rate have an average length of at least 2 mm, preferably at least

[0204] 3 mm, more preferably at least 4 mm, even more preferably at least 5 mm or even more than that. A method of producing a protein, such as a mycoprotein and / or mycelium fibers, preferably mycelium, in a bioreactor (10), the bioreactor (10) having a reactor body (12) with a reactor wall (14), the reactor wall (14) confining a reactor volume for incubating an inoculum configured for producing the protein, the reactor wall (14) having a tapering bottom section (16), a bottom opening (18) provided in the reactor wall (14) at the tapering bottom section (16), and a cap (20) for closing the bottom opening (18), the cap (20) including a top face (50), and a first fluid channel (36) extending through the cap (20), wherein the first fluid channel (36) is configured for transferring fluid between an inside of the reactor body (12) and an outside of the reactor body (12), and wherein an exit opening (46) of the first fluid channel (36) is arranged flush with the top face (50) of the cap (20), the method comprising the steps of: incubating the inoculum within the reactor body (12), and aerating the inoculum by providing air and / or oxygen through the first fluid channel (36) of the cap (20). The method of aspect 30, comprising the step of: feeding cultivation medium into the reactor body (12), preferably wherein the cultivation medium is fed through a lid (26, 68) closing a top opening of the reactor body (12), and / or through a second fluid channel (38) arranged in the cap (20), and aerating a mixture of inoculum and cultivation medium by providing air and / or oxygen through the first fluid channel (36) of the cap (20). The method of aspect 31, comprising the step of: continuously aerating the mixture of inoculum and cultivation medium for enabling growth of proteins, such as fungal mycelia, within the bioreactor (10). The method of any one of aspects 30 to 32, wherein aerating is such that air and / or oxygen bubbles are produced at the exit opening (46) of the first fluid channel (36) for stirring the mixture. The method of aspect 33, wherein aerating includes providing air and / or oxygen bubbles of at least 5 mm, preferably at least 8 mm, more preferably at least 10 mm, more preferably at least 20 mm, even more preferably at least 30 mm, at the exit opening (46) of the first fluid channel (36) for stirring the mixture. The method of any one of aspects 30-34, wherein aerating includes providing an air lift or bubble column within the reactor volume, preferably for creating a stir and more preferably a recirculation zone within the reactor volume. The method of any one of aspects 30-35, further comprising the step of providing additional aeration within the reactor volume using a sparger (82). The method of aspect 36, wherein the step of providing additional aeration includes providing air and / or oxygen bubbles having a size of less than 10 mm, preferably less than 5 mm, more preferably less than 2 mm, more preferably less than 1 mm; and / or in a range between about 0.1 mm and about 4 mm, such as in a range between about 0.1 mm and about 1 mm. The method of aspect 36 or aspect 37, wherein the additional aeration is provided at the tapered bottom section (16) of the reactor wall (14) and / or within the cap (20), preferably within at least one of the first and second fluid channel (36, 38), more preferably within the first fluid channel (36). The method of any one of aspects 30-38, wherein a concentration of oxygen in the air used for aeration is increased when compared to an oxygen level in ambient air to enhance cell growth; and / or wherein a concentration of oxygen in the air used for aeration is at least 22%, preferably at least 23%, more preferably at least 25%. The method of any one of aspects 31 to 39, wherein the bioreactor (10) is operated in fed-batch mode with the amount of cultivation medium in the bioreactor (10) being increased over a predetermined amount of time, preferably wherein the amount of medium is increased over a time of at least 5 hours, more preferably at least 12 hours, even more preferably at least 24 hours, preferably wherein the time is 48 hours or less, more preferably wherein the time is 30 hours or less. The method of any one of aspects 31 to 40, wherein the cultivation medium is fed such that exponential growth is maintained during feeding of the medium. The method of any one of aspects 31 to 41, further comprising: harvesting produced proteins, such as produced mycoproteins / fungal mycelium, from the bioreactor (10), preferably wherein harvesting is done through a second fluid channel (38) arranged in the cap (20), preferably wherein the second fluid channel (38) has a diameter (64) of at least 10 mm, more preferably at least 15 mm, to prevent clogging during harvesting. The method of aspect 42, further comprising: harvesting not more than a predetermined percentage of produced proteins or mycoproteins / fungal mycelium, such as harvesting between 60% and 95%, preferably 80% and 95%, and after harvesting, feeding further cultivation medium into the reactor body (12) for producing further protein within the same bioreactor (10). The method of any one of aspects 30 to 43, wherein inoculum is provided through a lid (26, 68) that is configured to close a top opening of the reactor body (12) and / or through a second or third fluid channel that is provided in the cap (20) closing the bottom opening (18) of the bioreactor (10). The method of any one of aspects 30 to 44, wherein the bioreactor (10) is operated in a continuous mode, the continuous mode comprising the steps of: harvesting a first portion of the produced protein, such as mycelium, while a second portion of the produced protein, such as mycelium, remains in the bioreactor, preferably in the reactor body; and feeding further cultivation medium into the reactor body and further incubating the second portion of the produced protein, such as mycelium, in the reactor body, preferably wherein harvesting and feeding is done essentially simultaneously or alternatingly, more preferably wherein harvesting and feeding is adapted such that cell growth is maintained during harvesting and feeding. The method of aspect 45, wherein the bioreactor (10) is operated in the continuous mode over a time period of 48 hours or more, preferably over a time period of 100 hours or more, more preferably over a time period of 500 hours or more, such as 800 hours or even longer. The method of any one of aspects 30 to 46, wherein the method includes producing mycelium fibers, such as mycoprotein fibers and / or hyphae,, having an average length of at least 2 mm , preferably at least 3 mm, more preferably at least 4 mm, even more preferably at least 5 mm, or even more than that. The method of any one of aspects 30-47, wherein the method includes producing mycelium fibers, such as mycoprotein fibers and / or hyphae, at a production rate of at least 10 g / L / d, preferably at least 15 g / L / d, more preferably at least 20 g / L / d, even more preferably at least 30 g / L / d .

Claims

CLAIMS1. A bioreactor (10) for producing a protein, such as a mycoprotein and / or mycelium fibers, the bioreactor (10) comprising: a reactor body (12) having a reactor wall (14), the reactor wall (14) confining a reactor volume for incubating an inoculum for producing the protein, the reactor wall (14) having a tapering bottom section (16), a bottom opening (18) provided in the reactor wall (14) at the tapering bottom section (16), and a cap (20) for covering the bottom opening (18), the cap (20) including a top face (50), and a first fluid channel (36) extending through the cap (20), wherein the first fluid channel (36) is configured for transferring fluid between an inside of the reactor body (12) and an outside of the reactor body (12), and wherein an exit opening (46) of the first fluid channel (36) is arranged flush with the top face (50) of the cap (20).

2. The bioreactor (10) of claim 1, wherein the top face (50) is planar, and / or is configured for providing a smooth transition to an inner surface of the reactor wall (14), and / or wherein, in a region around the bottom opening (18), the top face (50) of the cap (20) and an inner surface of the reactor wall (14) are arranged flush to one another.

3. The bioreactor (10) of claims 1 or 2, wherein the reactor body (12) comprises a cylindrical body section, wherein the cylindrical body section and / or the tapering bottom section (16) have a main axis (42), the first fluid channel (36) has a first fluid channel axis (40), and the first fluid channel axis (40) has an angled orientation to the main axis (42), preferably wherein an angle (44) between the main axis (42) and the first fluid channel axis (40) is in a range between 10° to 60°, more preferably between 20° to 30°, and / or an angle (44) between the main axis (42) and the first fluid channel axis (40) is adapted for creating a fluid swirl within the reactor volume.

4. The bioreactor (10) of any of claims 1 to 3, wherein the exit opening (46) is configured for producing air and / or oxygen bubbles having a size of at least 5 mm, preferably at least 8 mm, more preferably at least 10 mm, more preferably at least 20 mm, even more preferably at least 30 mm.

5. The bioreactor (10) of any of claims 1 to 4, wherein the exit opening (46) is delimited by an inner wall of the first fluid channel (36), preferably wherein the exit opening (46) has a diameter of at least 10 mm, preferably at least 15 mm, more preferably at least 20 mm, more preferably at least 25 mm.

6. The bioreactor (10) of any one of the previous claims, wherein the first fluid channel (36) includes a plurality of exit openings (46), each exit opening (46) being arranged flush with the top face (50) of the cap (20).

7. The bioreactor (10) of any one of claims 4-6, wherein the first fluid channel (36) includes a main channel portion (56) and a ring channel portion (58), and the ring channel portion (58) fluidly connects the main channel portion (56) with the plurality of exit openings (46).

8. The bioreactor (10) of any one of claims 4-7, wherein the exit openings (46) are arranged in a predetermined pattern on the top face (50) of the cap (20), preferably wherein the predetermined pattern is a circular pattern around an imaginary central point; and / or wherein the exit openings (46) extend at exit opening axes (60) that are oblique to the main axis (42), preferably wherein the exit opening axes (60) are arranged at an angle (62) to the main axis (42), the angle (62) being in a range between 10° to 60°, more preferably between 20° to 30°, more preferably wherein the angle (62) is configured to create a fluid swirl within the reactor volume.

9. The bioreactor (10) of any one of the preceding claims, wherein the reactor volume is free of a stirring device, such as a stirring shaft with stirring blades.

10. The bioreactor (10) of any one of the preceding claims, wherein the cap (20) includes a second fluid channel (38) that is separate from the first fluid channel (36), the second fluid channel (38) extending through the cap (20) and being configured for transferring fluid between the inside and the outside of the reactor body (12), and wherein an exit opening (48) of the second fluid channel (38) is arranged flush with the top face (50) of the cap (20).

11. The bioreactor (10) of claim 10, wherein the second fluid channel (38) is configured for discharging proteins and / or a broth including proteins, such as mycoproteins, from the reactor body (12), and / or the second fluid channel (38) aligns in parallel with a central or main axis (42) of the reactor body (12), and / or the first fluid channel (36) is configured for aerating a mixture of inoculum and a cultivation medium in the reactor volume, and / or the second fluid channel (38) has a diameter (64) of at least 10 mm, more preferably at least 15 mm, and / or33the second fluid channel (38) has a diameter (64) of 15 to 20 mm or larger, such as 32 mm or even larger, and / or a diameter (64) of the second fluid channel (38) is larger than a diameter of the first fluid channel (36) and / or an exit opening (46) of the first fluid channel (36), and / or a diameter (64) of the second fluid channel (38) is by a factor of 4, 6, 8, 10 or more, larger than a diameter of the first fluid channel (36) and / or an exit opening (46) of the first fluid channel (36).

12. The bioreactor (10) of any one of claims 10 or 11 in combination with any one of claims 4-8, wherein the exit openings (46) of the first fluid channel (36) are arranged in a predetermined pattern around the exit opening (48) of the second fluid channel (38), and / or the exit openings (46) of the first fluid channel (36) are arranged in a circular pattern and / or in a concentric manner around the exit opening (48) of the second fluid channel (38).

13. The bioreactor (10) of any one of the preceding claims, further comprising: a top opening provided in the reactor wall (14) and being configured for providing access to the reactor volume, and a lid (26, 68) for closing the top opening, the lid (26, 68) including a lid body with a channel (72) for transferring fluid between the inside and the outside of the reactor body (12), and at least one connection port (28, 70) fluidly connected to the channel, wherein the at least one connection port (28, 70) is configured for connecting to a respective fluid line for transferring fluid into or out of the reactor body, and / or is configured for connecting to a probe (30).

14. The bioreactor (10) of any one of the preceding claims, wherein the bioreactor (10) is adapted for fermentation, preferably submerged fermentation, more preferably for submerged fermentation of microbial protein / fungal mycelia used as food analogue, food stuff or food alternative, such as mycoprotein-based meat, fish, diary, seafood etc.

15. The bioreactor (10) of any one of the preceding claims, further comprising a sparger (82), preferably an air sparger, more preferably an oxygen sparger, for supplying air, preferably oxygen, to the reactor volume.

16. The bioreactor (10) of claim 15, wherein the sparger (82) includes one or more openings configured for producing bubbles, such as air and / or oxygen bubbles, the bubbles having a size of less than 5 mm, more preferably less than 4 mm, more preferably less than 2 mm, more preferably less than 1 mm.

17. The bioreactor (10) of claim 15 or claim 16, wherein the sparger (82) is arranged in the tapering bottom (16) section of the reactor wall (14).

18. The bioreactor (10) of claim 15 or claim 16, wherein the sparger (82) is arranged within the cap (20).

19. The bioreactor (10) of claim 18, preferably in combination with any one of aspect 12-14, wherein the sparger (82) is arranged within at least one of the first fluid channel (36) and the second fluid channel (38), preferably within the first fluid channel (36).

20. The bioreactor (10) of any one of the preceding claims, wherein the bioreactor (10) is configured for producing mycelium fibers, such as mycoprotein fibers and / or hyphae, having an average length of at least 2 mm, preferably at least 3 mm, more preferably at least 4 mm, even more preferably at least 5 mm, or even more than that.

21. The bioreactor (10) of any one of the preceding claims, wherein the bioreactor (10) is configured for producing mycelium fibers, such as mycoprotein fibers and / or hyphae with a production rate of at least 10 g / L / d, preferably at least 15 g / L / d, more preferably at least 20 g / L / d, even more preferably at least 30 g / L / d, preferably wherein the mycelium fibers, such as mycoprotein fibers and / or hyphae, produced with said production rate have an average length of at least 2 mm, preferably at least 3 mm, more preferably at least 4 mm, even more preferably at least 5 mm, or even more than that.

22. A method of producing a protein, such as a mycoprotein and / or mycelium fibers, preferably mycelium, in a bioreactor (10), the bioreactor (10) having a reactor body (12) with a reactor wall (14), the reactor wall (14) confining a reactor volume for incubating an inoculum configured for producing the protein, the reactor wall (14) having a tapering bottom section (16), a bottom opening (18) provided in the reactor wall (14) at the tapering bottom section (16), and a cap (20) for closing the bottom opening (18), the cap (20) including a top face (50), and a first fluid channel (36) extending through the cap (20), wherein the first fluid channel (36) is configured for transferring fluid between an inside of the reactor body (12) and an outside of the reactor body (12), and wherein an exit opening (46) of the first fluid channel (36) is arranged flush with the top face (50) of the cap (20), the method comprising the steps of: incubating the inoculum within the reactor body (12), and aerating the inoculum by providing air and / or oxygen through the first fluid channel (36) of the cap (20).

23. The method of claim 22, wherein aerating includes providing air and / or oxygen bubbles of at least 5 mm, preferably at least 8 mm, more preferably at least 10 mm at the exit opening (46) of the first fluid channel (36) for stirring the mixture.

24. The method of claim 22 or claim 23, wherein aerating includes providing an air lift or bubble column within the reactor volume, preferably for creating a stir and more preferably a recirculation zone within the reactor volume.

25. The method of any one of claims 22-24, further comprising the step of providing additional aeration within the reactor volume using a sparger (82).

26. The method of claim 25, wherein the step of providing additional aeration includes providing air and / or oxygen bubbles having a size of less than 5 mm, more preferably less than 4 mm, more preferably less than 2 mm, more preferably less than 1 mm.

27. The method of claims 25 or claim 26, wherein the additional aeration is provided at the tapered bottom section (16) of the reactor wall (14) and / or within the cap (20), preferably within at least one of the first and second fluid channel (36, 38), more preferably within the first fluid channel (36).

28. The method of any one of claims 22-27, comprising the step of: feeding cultivation medium into the reactor body (12), preferably wherein the cultivation medium is fed through a lid (26, 68) closing a top opening of the reactor body (12), and / or through a second fluid channel (38) arranged in the cap (20), and aerating a mixture of inoculum and cultivation medium by providing air and / or oxygen through the first fluid channel (36) of the cap (20), preferably continuously aerating the mixture of inoculum and cultivation medium for enabling growth of fungal mycelia, within the bioreactor (10).

29. The method of any one of claims 22-28, wherein the bioreactor (10) is operated in fed-batch mode with the amount of cultivation medium in the bioreactor (10) being increased over a predetermined amount of time, preferably wherein the amount of medium is increased over a time of at least 5 hours, more preferably at least 12 hours, even more preferably at least 24 hours, preferably wherein the time is 48 hours or less, more preferably wherein the time is 30 hours or less, and / or the cultivation medium is fed such that exponential growth is maintained during feeding of the medium, and / or not more than a predetermined percentage of produced proteins, such as between 60% and 95%, preferably 80% and 95%, is harvested, and after harvesting, further cultivation medium into the reactor body (12) is fed for producing further protein within the same bioreactor (10).

30. The method of any one of claims 22-29, wherein the bioreactor (10) is operated in a continuous mode, the continuous mode comprising the steps of:36harvesting a first portion of the produced protein while a second portion of the produced protein remains in the bioreactor, preferably in the reactor body; and feeding further cultivation medium into the reactor body and further incubating the second portion of the produced protein in the reactor body, preferably wherein harvesting and feeding is done essentially simultaneously or alternatingly, more preferably wherein harvesting and feeding is adapted such that cell growth is maintained during harvesting and feeding.37

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