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WO2025068592A2 - Système de bioréacteur et procédé de production agricole d'aliments fermentés - Google Patents

Système de bioréacteur et procédé de production agricole d'aliments fermentés Download PDF

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Publication number
WO2025068592A2
WO2025068592A2 PCT/EP2024/077443 EP2024077443W WO2025068592A2 WO 2025068592 A2 WO2025068592 A2 WO 2025068592A2 EP 2024077443 W EP2024077443 W EP 2024077443W WO 2025068592 A2 WO2025068592 A2 WO 2025068592A2
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WO
WIPO (PCT)
Prior art keywords
reactor vessel
vessel
fermented feed
fermentation
bioreactor system
Prior art date
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PCT/EP2024/077443
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English (en)
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WO2025068592A3 (fr
Inventor
Lidia García POU
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Dapibus AB
Original Assignee
Dapibus AB
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Publication date
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Publication of WO2025068592A2 publication Critical patent/WO2025068592A2/fr
Publication of WO2025068592A3 publication Critical patent/WO2025068592A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/58—Reaction vessels connected in series or in parallel
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/32—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of substances in solution
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements

Definitions

  • the present disclosure relates to a bioreactor system for agricultural production of fermented feed.
  • the system can be installed at a farm to produce feed locally.
  • the disclosure further relates to method for production of fermented feed.
  • Fermentation is a metabolic process where microorganisms, such as bacteria, yeast, or fungi, convert organic compounds, typically carbohydrates, into other compounds. The process is widely used in the production of various food.
  • the growth medium may be pasteurized milk and the microorganism culture may comprise lactic acid bacteria.
  • the temperature needs to be controlled to create a favorable environment for the lactic acid bacteria.
  • a number of further parameters may typically be monitored and/or controlled during the process.
  • Production of fermented feed for animals, on farms and in general, has several advantages, including, for example, improved digestibility, nutrient enrichment and probiotic effects on the animals’ gut bacteria. Dietary manipulation may not only positively affect the growth and health of the animals on the farms, but may also play a role in reducing productions costs and pollution, directly and indirectly.
  • a bioreactor is a device or vessel for the growth and cultivation of various biological organisms under controlled conditions. These controlled conditions typically include parameters like temperature, pH, agitation, aeration, nutrient supply, and monitoring of various process variables.
  • a few systems for production of fermented feed have previously been described.
  • production is usually carried out at production facilities, from which the produced feed is then shipped to the farmers. Since farmers often have feedstock that can be used in the production of fermented feed available at the farm, it would be beneficial to be able to produce the fermented feed locally at the farm rather than first transporting the feedstock to the production facilities and then transport the produced fermented feed back to the farm.
  • the known systems for batch or continuous production come with a number of known challenges and inconveniences.
  • the process in a batch reactor is simpler to carry out but once it has finished the farmer would need to clean the system and obtain new microorganism culture for every batch.
  • a process in a continuous reactor has the advantage that it can, at least theoretically, continue for a long time, but is significantly more difficult to control and may often have side effects and can produce undesired microorganisms after some time.
  • It is an object of the present disclosure is to provide a bioreactor system that can be used locally at a farm for agricultural production of fermented feed.
  • the bioreactor system provides improvements regarding the production capacity and reduced need for new microorganism culture, in particular in relation to the simplicity and ease of use that it offers.
  • a bioreactor system for agricultural production of fermented feed comprising: a first reactor vessel comprising: one or more first vessel inlets for receiving feedstock, water and microorganism culture; a first vessel outlet for removing first batch fermented feed from the first reactor vessel; a second reactor vessel fluidly connected to the first reactor vessel; one or more second vessel inlets for receiving feedstock and water; a second vessel fermented feed inlet for receiving fermented feed from the first reactor vessel; a second vessel outlet for removing second batch fermented feed from the second reactor vessel; a sensor for sensing a level of fermentation in at least the first reactor vessel, wherein the bioreactor system is a closed and/or aseptic bioreactor system; a control unit for controlling the agricultural production of fermented feed in the first reactor vessel and the second reactor vessel, wherein the control unit is configured to: receive measurements from the sensor for sensing a level of fermentation in the first reactor vessel; control a valve between the first reactor vessel and the at least one second vessel
  • the inventors have realized that by using at least two serially connected reactor vessels in a closed and/or aseptic system, and setting up a process in which a portion of the produced fermented feed from the first reactor vessel is used as microorganism culture in the second reactor vessel, a system is provided, which has properties that are particularly useful for local operation at a farm.
  • the bioreactor system has several advantages.
  • the processes in the first reactor vessel and the second reactor vessel are batch processes i.e. relatively straightforward processes to control, at least in relation to a continuous process. At the same time production can be extended without the need for more microorganism culture and spread out over a longer period of time.
  • the process in the second reactor vessel starts when the process in the first reactor vessel stops. Since the processes are substantially sequential, possibly partly overlapping, some parts that are used for the processes (heater, cooler, sensors, members for circulating content etc.) can optionally be shared between the two or more reactor vessels.
  • the presently disclosed setup has turned out to be particularly advantageous as a local system installed at a farm.
  • feedstock available that is suitable for production of fermented feed. Examples include, but are not limited to, oat, potato, sugar beets, barley, wheat and hay.
  • the microorganism culture is typically more complex for the farmer to provide, and therefore this part may typically be obtained from an external source.
  • the presently disclosed system and method allows the local farmer to use less externally obtained microorganism culture.
  • the present disclosure further relates to a method for production of fermented feed, the method comprising: providing a bioreactor system comprising: a first reactor vessel comprising a first vessel outlet for removing first batch fermented feed from the first reactor vessel; a second reactor vessel fluidly connected to the first reactor vessel, the second reactor vessel comprising a vessel fermented feed inlet for receiving first batch fermented feed from the first reactor vessel; filling the first reactor vessel with water and feedstock; adding a microorganism culture to the first reactor vessel, wherein the microorganism culture comprises yeast and/or fungus; controlling a fermentation process in the first reactor vessel; when a level of fermentation has reached a predetermined level of fermentation, transferring a seed train part of a first batch fermented feed from the first reactor vessel to the second reactor vessel through the vessel fermented feed inlet of the second reactor vessel; emptying a remaining production part of the first batch fermented feed from the first reactor vessel; controlling a fermentation process in the second reactor vessel; when a level of fermentation has reached
  • FIG. 1 shows an embodiment of the presently disclosed bioreactor system for agricultural production of fermented feed.
  • Fig. 2 shows a conceptual example of the presently disclosed bioreactor system for agricultural production of fermented feed with possible flow of feedstock, water, microorganism culture and fermented feed.
  • Fig. 3 shows an embodiment of a disinfection channel.
  • Fig. 4 shows a flowchart of a method according to an embodiment of the presently disclosed method for production of fermented feed.
  • Fig. 5 shows a further embodiment of the presently disclosed bioreactor system for agricultural production of fermented feed.
  • the present disclosure relates to a bioreactor system for agricultural production of fermented feed.
  • the system comprises at least a first reactor vessel and a second reactor vessel.
  • the system is not limited to two reactor vessels - it may comprise further reactor vessels, such as a third reactor vessel, a fourth reactor vessel and so forth.
  • one embodiment of the bioreactor system further comprises at least one further third reactor vessel fluidly connected to the first reactor vessel and/or the second reactor vessel; one or more third vessel inlets for receiving feedstock and water; a third vessel fermented feed inlet for receiving fermented feed from the first and/or second reactor vessel; and a third vessel outlet for removing third batch fermented feed from the third reactor vessel.
  • the system may further comprise a fourth reactor vessel, a fifth reactor vessel etc. with the same features.
  • the first reactor vessel comprises one or more first vessel inlets for receiving feedstock, water and microorganism culture. This means that there may, for example, be one inlet for receiving feedstock and water and another inlet for receiving the microorganism culture.
  • the first reactor vessel is fluidly connected to the second reactor vessel.
  • the first reactor vessel also has a first vessel outlet for removing first batch fermented feed from the first reactor vessel.
  • the second reactor vessel comprises one or more second vessel inlets for receiving feedstock and water.
  • the second reactor vessel further comprises a second vessel fermented feed inlet for receiving fermented feed from the first reactor vessel.
  • the second vessel fermented feed inlet can be connected to the first vessel outlet or to a separate connection or outlet for the seed train part of the first batch fermented feed.
  • the bioreactor system described herein is a closed and/or aseptic bioreactor system designed to prevent contamination and ensure sterile conditions throughout the fermentation process.
  • the system maintains a controlled, sealed environment to allow for precise management of microbial activity, ensuring product purity and consistency.
  • the first reactor vessel and the second reactor vessel may be fully enclosed and configured to operate in a sealed manner. All inlets and outlets may be equipped with sterile seals, gaskets, or aseptic connectors to prevent the introduction of external contaminants.
  • the vessels may be constructed from non-reactive, sterilizable materials, such as stainless steel or biocompatible polymers, that resist microbial infiltration.
  • Each vessel may include one or more aseptic inlets for the addition of feedstock, water, and microorganism culture. These inlets may be further equipped with sterile filters to ensure that no airborne microorganisms or particles enter the system during material transfer. Similarly, aseptic outlets allow for the removal of the fermented product while maintaining sterility within the vessel. These outlets may be connected to a sterile transfer system that moves the product to downstream processing without exposure to the external environment.
  • the system may include aseptic sampling ports for taking samples of the fermentation medium without introducing contaminants.
  • Such ports are designed with sterile connections and barriers that allow for sampling while maintaining the closed environment.
  • Sensors such as for pH, temperature, and dissolved oxygen, may be integrated into the system using sterile connectors to monitor critical fermentation parameters in real time without compromising system sterility.
  • the microorganism culture may be introduced into the first reactor vessel through an aseptic feed system.
  • the system may include a sterile reservoir for the microorganism culture, which is connected to the reactor vessel via sterile tubing and aseptic fittings.
  • the system may include a filtered gas exchange system that allows air or oxygen to be introduced into the reactor while maintaining sterility.
  • the bioreactor system may be configured to operate under controlled pressure conditions to maintain its sealed nature.
  • Pressure relief valves may be included, which are fitted with sterile filters to allow for pressure regulation without compromising the aseptic integrity of the system.
  • the bioreactor system may be configured to operate as a closed loop, meaning no external exposure occurs at any stage. This ensures that the system remains fully aseptic from the start of fermentation to the final product removal.
  • the microorganism culture may comprise yeast and/or fungus. These microorganisms are selected for their ability to efficiently metabolize the feedstock and produce the desired fermentation by-products, such as organic acids, ethanol, or other valuable compounds.
  • Yeast which are unicellular fungi, are commonly used in fermentation processes due to their high metabolic activity and ability to convert sugars into ethanol and carbon dioxide.
  • Yeast strains such as Saccharomyces cerevisiae, may be particularly advantageous in processes where rapid fermentation of sugars is required, as they are highly efficient in breaking down carbohydrates under both aerobic and anaerobic conditions.
  • the microorganism culture may comprise fungus, which includes a broader group of eukaryotic organisms that can exist in unicellular or multicellular forms.
  • Fungi are particularly useful in solid-state fermentation or in processes that involve the breakdown of complex organic substrates such as cellulose, lignin, and other plant-derived materials.
  • filamentous fungi such as Aspergillus or Rhizopus species can be employed in processes requiring enzymatic breakdown of more complex carbohydrates into simpler sugars, which can then be further fermented.
  • the microorganism culture may include both yeast and fungus, allowing for a synergistic fermentation process where each organism performs complementary roles.
  • yeast may ferment simple sugars into ethanol, while the fungus breaks down more complex substrates, thereby increasing the overall efficiency of the fermentation process.
  • the microorganism culture may be introduced into the first reactor vessel under sterile conditions, ensuring that the fermentation proceeds without contamination.
  • the operating conditions within the reactor such as temperature, pH, and oxygen levels, can be adjusted to optimize microbial growth and fermentation activity.
  • the bioreactor system may include specific design features to ensure it can support the unique growth and metabolic requirements of these microorganisms.
  • Yeast which are unicellular fungi, may be used in either solid state fermentation or using liquid.
  • the bioreactor may include an agitation system, such as impellers or stirrers, that provides adequate mixing without damaging the yeast cells in case of the use of liquid.
  • Filamentous fungi which form delicate mycelial networks, typically require more gentle agitation to avoid damaging the hyphae.
  • the bioreactor system may therefore include adjustable agitation settings to accommodate both the vigorous mixing needed for yeast and the lower-shear mixing required for fungi.
  • Oxygen control is another feature, as different microorganisms have varying oxygen requirements.
  • Yeast strains such as Saccharomyces cerevisiae can operate under both aerobic and anaerobic conditions, so the bioreactor system may have a flexible oxygen control system that can adjust oxygen levels accordingly.
  • the bioreactor may therefore include a gas exchange system with sterile filters to ensure that sufficient oxygen is provided for fungal growth while maintaining sterility.
  • Temperature control may be included as well, as both yeast and fungi require precise temperature conditions for optimal fermentation. Yeast generally perform well at temperatures between 20°C and 35°C, while fungi may require different temperature ranges depending on the species.
  • the bioreactor system may be equipped with heating and cooling capabilities to maintain the appropriate temperature for both types of microorganisms.
  • the bioreactor may further comprise a pH monitoring and control system, as fermentation can cause significant pH changes due to the production of organic acids and other by-products.
  • Yeast typically require a pH range of 4.0 to 6.0, while fungi may have slightly different pH requirements.
  • An automatic system for adding acid or base may be included to maintain the optimal pH for the fermentation process.
  • the bioreactor may be designed to handle processes where the substrate is in a semi-solid or solid state with minimal free-flowing liquid.
  • the system may therefore comprise air distribution and humidity control to ensure fungal growth on solid substrates.
  • the bioreactor system may therefor include clean-in-place (CIP) and steam-in-place (SIP) systems to sterilize the vessels, pipes, and inlets before and after fermentation. Additionally, all inlets and outlets may be be sealed with sterile filters to prevent contamination during operation.
  • CIP clean-in-place
  • SIP steam-in-place
  • the second vessel fermented feed inlet may be connected to a corresponding fermented feed outlet of the first reactor vessel, wherein the fermented feed outlet of the first reactor vessel, the second vessel fermented feed inlet and a connection between them form a closed connection.
  • This connection may be designed as a closed system, ensuring that the transfer of fermented feed from the first vessel to the second occurs without exposure to the external environment, thereby maintaining the aseptic conditions of the system.
  • the fermented feed outlet of the first reactor vessel may be configured to transfer a portion of the fermented material to the second reactor vessel.
  • This outlet is preferably securely connected to the fermented feed inlet of the second reactor vessel, allowing for the controlled and sterile movement of the fermented feed.
  • the connection between these two vessels forms a closed connection, which may include sterile tubing or piping with appropriate gaskets, seals, or sterile quick-connect fittings to ensure no contamination can occur during the transfer process.
  • the bioreactor system may be a solid state bioreactor system.
  • the bioreactor system may be configured to support fermentation processes where the substrate is in a solid or semi-solid state, with minimal free-flowing liquid.
  • the system may therefore include controlled air flow or forced aeration to ensure adequate oxygen levels for the microorganisms.
  • Effective temperature control may be included because microbial activity in solid-state fermentation generates heat, which can accumulate and affect the fermentation process.
  • the bioreactor typically includes cooling systems, such as air cooling or heat exchangers, to maintain optimal temperature conditions.
  • Agitation may or may not be present depending on the design. While some solid-state bioreactors are static and do not involve agitation, others include mechanical agitation or stirring mechanisms to prevent clumping and ensure uniform temperature and microbial distribution. For systems that include agitation, the mixing is generally gentle to avoid damaging the solid matrix or microorganisms.
  • Solid-state bioreactors offer a large surface area for microbial growth, which is important for microorganisms like fungi that grow on the surface of solid substrates. This allows for efficient metabolism and fermentation.
  • Humidity control is another factor, as solid-state fermentation may require moisture management.
  • Some bioreactors incorporate humidifiers or moisture control systems to maintain the optimal level of humidity without introducing excess free water.
  • the system is also be designed to handle by-products of the fermentation process, such as gases or liquids produced by microbial metabolism. It may include venting mechanisms for gas release and drainage systems to manage any liquid that may accumulate during fermentation.
  • Solid-state bioreactors are particularly suited for fungi and certain bacteria that grow well in environments with low water activity, making them ideal for the production of enzymes, organic acids, and other valuable products. These systems may be either static, where the substrate remains stationary, or rotating, where the substrate is tumbled or gently mixed to distribute microorganisms evenly. In both cases, the bioreactor design may be designed to maintain sterility to prevent contamination, which can be achieved through sterile inlets, outlets, filters, and sometimes clean-in-place or steam-in-place systems.
  • Each of the first reactor vessel and the second reactor vessel may have a volume of a least 200 liters, but may have a volume of a least 500 liters, 1000 liters or at least 2000 liters.
  • the system further comprises a sensor for sensing a level of fermentation in at least the first reactor vessel.
  • the system may further comprise a number of additional sensors for various purposes.
  • the system further comprises a control unit for controlling the agricultural production of fermented feed in the first reactor vessel and the second reactor vessel. The control unit receives measurements from the sensor for sensing a level of fermentation in at least the first reactor vessel.
  • the control unit may control a valve between the first reactor vessel and the at least one second vessel fermented feed inlet of the second reactor vessel to transfer a seed train part of the first batch fermented feed from the first reactor vessel to the second reactor vessel through the second vessel fermented feed inlet.
  • a ‘valve’ shall be interpreted broadly to comprise any suitable control mechanism for regulating that the seed train part of the first batch fermented feed is transferred to the second reactor vessel.
  • the control unit may also control the process of removing the rest of the first batch fermented feed from the first reactor vessel.
  • the sequence of obtaining a first batch feed from the first reactor vessel and transferring a seed train part of it to the second reactor vessel may be done in any order.
  • fermentation in the first reactor vessel is deactivated when the seed train part of the first batch fermented feed has been transferred from the first reactor vessel to the second reactor vessel.
  • the rest of the first batch fermented feed can then be removed from the first reactor vessel.
  • Fig. 1 shows an example of an embodiment of the presently disclosed bioreactor system 100 for agricultural production of fermented feed.
  • the system 100 comprises a first reactor vessel 101 and a second reactor vessel 102.
  • the first reactor vessel 101 has a first vessel inlet 103 and a first vessel outlet 104.
  • the second reactor vessel 102 has a second vessel inlet 105, a second vessel outlet 107 and a second vessel fermented feed inlet 106.
  • the system comprises a control unit 108 for controlling the processes in the first reactor vessel 101 and a second reactor vessel 102.
  • There is a valve 111 for controlling the transfer of the seed train part of the first batch fermented feed from the first reactor vessel 101 to the second reactor vessel 102.
  • the second vessel fermented feed inlet 106 could also have been connected to the first vessel outlet 104.
  • the embodiment further comprises air inlets 117 for sterile air supply.
  • each of the first reactor vessel 101 and the second reactor vessel 102 has a heater 113 and a mixer 114. It would be possible to share these components.
  • the bioreactor system 100 further comprises a sensor for sensing a level of fermentation 109, a first volume sensor 110, a second volume sensor 112, a cooler 115, a third volume sensor 118 and a fourth volume sensor 119.
  • Fig. 5 shows a further embodiment of the presently disclosed bioreactor system 100 for agricultural production of fermented feed.
  • the system 100 comprises a first reactor vessel 101 and a second reactor vessel 102.
  • the first reactor vessel 101 has a first vessel inlet 103 and a first vessel outlet 104.
  • the second reactor vessel 102 has a second vessel inlet 105, a second vessel outlet 107 and a second vessel fermented feed inlet 106.
  • the system comprises a control unit 108 for controlling the processes in the first reactor vessel 101 and a second reactor vessel 102.
  • There is a valve 111 for controlling the transfer of the seed train part of the first batch fermented feed from the first reactor vessel 101 to the second reactor vessel 102.
  • the second vessel fermented feed inlet 106 could also have been connected to the first vessel outlet 104.
  • the embodiment further comprises air inlets 117 for sterile air supply.
  • each of the first reactor vessel 101 and the second reactor vessel 102 has a heater 113 and a mixer 114. It would be possible to share these components.
  • the bioreactor system 100 further comprises a sensor 109 for sensing a level of fermentation, a first volume sensor 110, a second volume sensor 112, a cooler 115, a third volume sensor 118 and a fourth volume sensor 119.
  • Fig. 2 shows a conceptual example of the presently disclosed bioreactor system 100 for agricultural production of fermented feed with possible flow of feedstock, water, microorganism culture and fermented feed.
  • the figure shows the flow of feedstock, water, microorganism culture, fermented feed seed train, fermented feed and sterile air for an example of the presently disclosed bioreactor system 100.
  • the concept may be applied to a first reactor vessel 101 , a second reactor vessel 102 and further reactor vessels, for example, a third reactor vessel 116.
  • the bioreactor system may be closed, preferably an aseptic, or at least highly hygienic, system.
  • the bioreactor system may further comprise one or more air inlets for supplying sterile air in the first reactor vessel and/or second reactor vessel. Supplying sterile air is a way of maintaining a clean environment, prevent contamination and make sure that the produced feed is of a certain quality. Non-sterile air could introduce variable factors and potentially disrupt the controlled conditions required for the fermentation process.
  • the bioreactor system may comprise one or more members or devices for stirring, mixing and/or circulating content of the first reactor vessel and/or second reactor vessel. All of these can either be shared by reactor vessels or local elements/devices for each reactor vessel.
  • the first step in the process may typically be preparation of the feedstock.
  • an additive can be added and grinding can be performed for obtaining a good feedstock to use in the fermentation process.
  • the bioreactor system may comprise at least one first volume sensor.
  • the control unit may stop the process of adding water and start adding feedstock to the first reactor vessel.
  • the bioreactor may further comprise at least one second volume sensor.
  • the control unit may stop the process of adding feedstock to the first reactor vessel.
  • the process of filling feedstock can be done in the same for the second reactor vessel. This is typically done later in the process, after the fermentation has been completed in the first reactor vessel, but the process can be the same i.e.
  • the bioreactor system may comprise at least one third volume sensor. When the third volume sensor detects that water has been filled to a predetermined water level, the control unit may stop the process of adding water and start adding feedstock to the second reactor vessel.
  • the bioreactor may further comprise at least one fourth volume sensor. When the fourth volume sensor detects that the water and feedstock have reached a predefined water and feedstock level, the control unit may stop the process of adding feedstock to the second reactor vessel.
  • the bioreactor may comprise one or more heaters.
  • the heater may be used in an initial heat sterilization process and/or in the process of controlling the slower fermentation process and/or for deactivation of the fermentation process.
  • the heater may function based on principles simple to those of common heating devices in various heating devices. It may comprise, for example, coils and/or wires.
  • the control unit may be configured to control the heater such that water and feedstock is heated for a predefined period of time upon having received water and feedstock. Still part of the process may be referred to as a heat sterilization process. In this process the water and feedstock may be heated for at least 10 minutes, preferably for at least one hour, more preferably at least two hours, at a temperature of at least 95°C, preferably at least 98°C. However, the process may depend on several parameters and variables, including the type of feedstock. A person skilled in the art would generally have the knowledge to select the parameters for a given type of feedstock.
  • the feedstock may then be cooled to a temperature suitable for the fermentation process.
  • the bioreactor status may, accordingly, comprise a cooling device or cooling mechanism, and the control unit may be configured to cool the water and feedstock after having heated the water and feedstock.
  • Common cooling mechanisms include refrigeration units, cooling coils and/or circulation of a cold fluid.
  • Active cooling may also be used if the fermentation process requires precise temperature control. In many fermentation processes it is useful to maintain the right temperature throughout the process. For example, the fermentation process itself may generate heat. In such cases it may be useful to control the temperature of the content to stay within a predetermined temperature range.
  • the cooler and the heater and the control unit
  • the control unit may be further configured to control a process of adding a microorganism culture to the first reactor vessel.
  • the process of adding the microorganism culture to the first reactor vessel is typically performed after having heated and optionally cooled the water and feedstock.
  • the microorganism culture may be added using the same inlet as the process of adding feedstock and water.
  • an aseptic barrier may be used to avoid introduction of contamination.
  • the inventors have realized that this can be achieved by a solution in which at least one of the one or more first vessel inlets for receiving microorganism culture is connected to a microorganism vessel through a disinfection connection.
  • the disinfection connection may comprise a first membrane towards the first reactor vessel, a second membrane towards the microorganism vessel and a disinfection compartment disposed between the first membrane and the second membrane.
  • the bioreactor system may comprise a sterile tank for the microorganism culture, and a pump system and/or a valve system for delivery of the microorganism culture to the first reactor vessel.
  • the sterile tank can be maintained under sterile conditions to prevent contamination of the culture.
  • system is further equipped with either a pump system, a valve system, or a combination of both, to ensure precise and controlled delivery of the microorganism culture to the first reactor vessel.
  • the pump system may use, for example, peristaltic or diaphragm pumps to transport the culture through sterile tubing, while the valve system allows for the regulated opening and closing of flow paths, ensuring that the microorganism culture is delivered at the appropriate time and in the correct volume, all while maintaining aseptic conditions.
  • the disinfection channel 121 comprises a first membrane 122 towards the first reactor vessel and a second membrane 123 towards the microorganism vessel (not visible in fig. 3 but shown in fig. 1) and a disinfection compartment 124 disposed between the first membrane 122 and the second membrane 123.
  • the disinfection channel 121 has one connection 125 to the first reactor vessel and one connection 126 to the microorganism vessel.
  • the connection 125 to the first reactor vessel comprises a pointed member 127, which can be used to puncture the one or both of the first membrane 122 and the second membrane 123.
  • the pointed member 127 comprises a pointed member channel 128, which allows a disinfectant in the disinfection compartment to disinfect the connection.
  • the bioreactor system may be configured such that one or more additional valves control for how long the disinfectant is applied to the connection, i.e. for how long it needs to stay in the connection and when the microorganism culture can be injected into the first reactor vessel.
  • control unit may be configured to control a fermentation process in the first reactor vessel upon having added a microorganism culture to the first reactor vessel.
  • a temperature control system and/or a mixing system may be used.
  • the temperature may typically be controlled in a closed-loop control system, which typically includes one or more temperature sensors and both or one of a heater and a cooler.
  • the fermentation process in the first reactor vessel is controlled by controlling the temperature in the first reactor vessel.
  • pH can be measured using one or more pH sensors and/or conductivity sensors and controlled using, for example, acid and base reservoirs and/or by using buffering agents.
  • the fermentation process can be considered done when the level of fermentation has reached a predetermined level of fermentation.
  • the level of fermentation may be defined as the amount of feedstock, such as the amount of carbohydrate, that has been consumed by the microorganisms, and/or as the amount of microorganisms that have been produced from the feedstock.
  • the level of fermentation can thus be measured in various ways, which would be known to the person skilled in the art.
  • the sensor for sensing a level of fermentation may comprise a conductivity sensor and/or a brix meter and/or an optical sensor and/or a density and/or biomass sensor and/or sensor for sensing suspended solid.
  • an optical sensor can measure the optical density of microbial mass concentration.
  • a brix meter can also be used to measure the progress of fermentation in some applications, in particular where sugar consumption or production of sugar is an indicator of fermentation progress.
  • the conductivity sensor may be configured to measure the ability to conduct electricity to determine a level of fermentation, and/or wherein the brix meter is configured to measure the brix value to determine a level of fermentation
  • This step of sensing a level of fermentation can be performed using various sensors and measurement techniques that monitor the progress and extent of fermentation by tracking specific variables associated with microbial activity and substrate conversion.
  • a pH sensor can continuously monitor changes in pH. When the pH drops to a specific threshold, it indicates that fermentation has progressed to a desired level.
  • a dissolved oxygen sensor or oxygen probe can measure the amount of oxygen remaining in the vessel.
  • a low DO level would signal that the microorganisms have been actively fermenting, which could be a sign that the process is near completion or has reached a target state.
  • a brix meter is used to measure the brix value, which represents the sugar content in a liquid solution.
  • the Brix value is expressed as the percentage of dissolved sugar in the liquid.
  • Brix sensors are commonly used in industries such as winemaking, brewing, and food production to monitor the fermentation process, as sugar is converted into alcohol, acids, or other by-products by microorganisms. Further sensors, such as biomass sensors or gas chromatography sensors, may be used.
  • Conductivity measurements are another effective method for monitoring the fermentation process in a bioreactor system. Electrical conductivity measures the ability of a solution to conduct electricity, which is influenced by the concentration of ions (such as salts, acids, and bases) in the solution. During fermentation, changes in the concentration of ions, as well as the production of metabolic by-products (such as organic acids), can cause shifts in the conductivity of the medium.
  • the same sensor, or an additional sensor, may be configured for sensing a level of fermentation in the second reactor vessel.
  • the fermentation process may be finished by another heating step, if necessary.
  • the fermentation does not necessarily have to reach a state in which the fermentation cannot continue anymore. It has turned out that it may be better to stop the fermentation when the level of fermentation has reached a predetermined level of fermentation selected from a range of 55-90% of a final state of fermentation, preferably selected from a range of 60-80% of a final state of fermentation.
  • the final state of fermentation may alternatively be expressed as a protein content.
  • the predetermined level of fermentation, or the stop criteria is selected such that the process is stopped when the protein content has reached a predetermined protein level selected from the range of 10-40% of the volume of the total content, preferably from the range of 15-32% of the volume of the total content.
  • a final state of fermentation may be defined as a state wherein all feedstock supplied to the reactor vessel has been consumed by the microorganisms, such as a state wherein all feedstock supplied to the reactor vessel has been converted into microorganisms.
  • a predetermined level of fermentation of 50% of a final state of fermentation may be defined as a state wherein 50% of the feedstock supplied to the reactor vessel has been consumed by the microorganisms, such as wherein 50% of the feedstock supplied to the reactor vessel has been converted into microorganisms.
  • the control unit may be configured to transfer the seed train part of the first batch fermented feed from the first reactor vessel to the second reactor vessel and empty a remaining production part of the first batch fermented feed from the first reactor vessel when the level of fermentation has reached the predetermined level of fermentation.
  • a valve between the first reactor vessel the second reactor vessel may control the transfer of the seed train part of the first batch fermented feed from the first reactor vessel.
  • the seed train part is the range 5-35% of the first batch fermented feed. Accordingly, the remaining production part of the first batch fermented feed may be in the range of 65-95% of the first batch fermented feed.
  • the production part of the first batch from the first reactor vessel and the second batch fermented feed from the second reactor vessel may be subject to further processing.
  • the bioreactor system may further comprise a mixer for mixing the production part of the first batch fermented feed and/or the second batch fermented feed with additional feed material, such as animal nutrition and/or acid.
  • the bioreactor system, or an additional system may also be adapted to, for example, dry or shape the production part of the first batch fermented feed and/or the second batch fermented feed to any suitable form.
  • the process carried out in the second reactor may, but does not necessarily have to, be carried out in the same way as the process in the first reactor vessel. This means that all technical details provided regarding the process in the first reactor vessel may be applied to the second reactor vessel. This also applies to possible further reactor vessels (third, fourth and so forth).
  • the control unit may thus be configured to control a fermentation process in the second reactor vessel upon having received the seed train part of the first batch fermented feed from the first reactor vessel. Moreover, the control unit may configured to deactivate fermentation in the second reactor vessel and empty a second batch fermented feed from the second reactor vessel when the level of fermentation has reached a second predetermined level of fermentation.
  • the present disclosure further relates to a method for production of fermented feed.
  • a bioreactor system according to any embodiment of the present disclosure is provided.
  • Such a bioreactor preferably comprises, as a minimum, a first reactor vessel comprising a first vessel outlet for removing first batch fermented feed from the first reactor vessel and a second reactor vessel fluidly connected to the first reactor vessel, the second reactor vessel comprising a vessel fermented feed inlet for receiving first batch fermented feed from the first reactor vessel.
  • the method may comprise a second step of filling the first reactor vessel with water and feedstock.
  • the method may further comprise a third step of adding a microorganism culture to the first reactor vessel.
  • the microorganism culture may comprise yeast and/or fungus.
  • the microorganism culture is added to the first reactor vessel through the disinfection connection described above.
  • the method may further comprise a fourth step of controlling a fermentation process in the first reactor vessel.
  • the method may further comprise a fifth step of transferring a seed train part of a first batch fermented feed from the first reactor vessel to the second reactor vessel through the vessel fermented feed inlet of the second reactor vessel when the level of fermentation has reached a predetermined level of fermentation. Fermentation in the first reactor vessel may then be deactivated.
  • the method may further comprise a sixth step of emptying a remaining production part of the first batch fermented feed from the first reactor vessel.
  • the method may further comprise a seventh step of controlling a fermentation process in the second reactor vessel.
  • a second predetermined level of fermentation the second level may be similar or the same as the level of fermentation used in the first reactor vessel
  • fermentation in the second reactor vessel may be deactivated.
  • a second batch fermented feed from the second reactor vessel can then be obtained.
  • the method may include the step of filling the first reactor vessel with water, which is then circulated throughout the vessel to ensure even distribution of heat. This circulation occurs while the water is heated to the desired temperature, effectively sterilizing the interior surfaces of the vessel.
  • the heating process continues for at least 10 minutes, though it is preferably conducted for a duration between 30 minutes and 2 hours, depending on the sterilization requirements. This step ensures that the vessel is thoroughly sanitized before the introduction of feedstock and microorganism culture, helping to maintain the aseptic conditions essential for the fermentation process.
  • the method may further comprise the step of cooling the water to a temperature between 25°C and 37°C, preferably to a temperature between 30°C and 35°C, after the water has been circulated and heated.
  • This cooling process ensures that the reactor vessel reaches the optimal temperature range for the subsequent introduction of the microorganism culture.
  • the controlled cooling maintains aseptic conditions while preparing the system for fermentation, ensuring that the microorganisms can thrive in a temperature environment conducive to their growth and activity.
  • the method may further include the step of adding the microorganism culture using an aseptic connection, ensuring that no contaminants are introduced during the process.
  • this is performed through a disinfection channel which comprises a first membrane positioned towards the first reactor vessel and a second membrane positioned towards the microorganism vessel. Between these two membranes is a disinfection compartment that ensures sterility during the transfer of the microorganism culture.
  • This configuration allows for the microorganism culture to be introduced into the first reactor vessel without compromising the sterile environment, as the disinfection channel acts as a barrier, preventing any contamination while facilitating a controlled and safe transfer.
  • the method may then further comprise the step of filling the second reactor vessel with water and feedstock, preparing it for the next stage of the fermentation process.
  • the method may further include the step of filling the first reactor vessel with pasteurized water from the second reactor vessel when the level of fermentation in the first reactor vessel has reached a predetermined level. This ensures that the water used in the first reactor vessel is sterile, maintaining the aseptic conditions required for continued fermentation.
  • the transfer of pasteurized water from the second reactor vessel helps streamline the process by utilizing existing resources within the system while ensuring proper sanitation and preparation for subsequent fermentation cycles.
  • the pasteurized water may then be transferred from the second reactor vessel to soak in the first reactor vessel for a time period between 30 seconds and 20 minutes.
  • This soaking period ensures proper hydration and thermal equilibrium within the vessel, further enhancing the sterility of the environment and ensuring that all internal surfaces are adequately treated before continuing with the fermentation process.
  • the duration of the soak can be adjusted depending on the specific requirements of the fermentation or preparation phase, ensuring optimal conditions for microbial activity once the microorganism culture is introduced.
  • the method may further comprise the step of subsequently transferring a mix of water and a seed train part of the first batch fermented feed from the first reactor vessel to the second reactor vessel. This transfer ensures that a portion of the active microorganism culture from the first reactor is used to inoculate the second reactor vessel, facilitating the continuation of the fermentation process.
  • the method promotes the even distribution of microorganisms and nutrients within the second reactor, optimizing the conditions for the next fermentation batch and ensuring process continuity.
  • the method may further comprise the step of deactivating the fermentation process in the first reactor vessel.
  • Fig. 4 shows a flowchart of a method according to an embodiment of the presently disclosed method for production of fermented feed (200).
  • the method in fig. 4 comprises the steps of: providing a bioreactor system (201); filling the first reactor vessel with water and feedstock (202); controlling a fermentation process in the first reactor vessel (203); transferring a seed train part of a first batch fermented feed from the first reactor vessel to the second reactor vessel (204); emptying a remaining production part of the first batch fermented feed from the first reactor vessel (205); controlling a fermentation process in the second reactor vessel (206); and emptying a second batch fermented feed from the second reactor vessel (207).
  • a bioreactor system for agricultural production of fermented feed comprising: a first reactor vessel comprising: one or more first vessel inlets for receiving feedstock, water and microorganism culture; a first vessel outlet for removing first batch fermented feed from the first reactor vessel; a second reactor vessel fluidly connected to the first reactor vessel; one or more second vessel inlets for receiving feedstock and water; a second vessel fermented feed inlet for receiving fermented feed from the first reactor vessel; a second vessel outlet for removing second batch fermented feed from the second reactor vessel; a sensor for sensing a level of fermentation in at least the first reactor vessel; a control unit for controlling the agricultural production of fermented feed in the first reactor vessel and the second reactor vessel, wherein the control unit is configured to: receive measurements from the sensor for sensing a level of fermentation in the first reactor vessel; control a valve between the first reactor vessel and the at least one second vessel fermented feed inlet of the second reactor vessel to transfer a seed train part of the first batch fermented feed from the first
  • bioreactor system according to item 1 , wherein the bioreactor system is a closed, preferably aseptic, system.
  • the bioreactor system according to any one of the preceding items, comprising at least one first volume sensor.
  • the control unit is configured to stop a process of adding water and start adding feedstock to the first reactor vessel when the water has reached a predefined water level.
  • control unit is configured to stop the process of adding feedstock to the first reactor vessel when the water and feedstock have reached a predefined water and feedstock level.
  • control unit is configured to control a process of heating the water and feedstock for a predefined period of time upon having received water and feedstock.
  • control unit is configured to control a process of heating the water and feedstock for at least one hour, preferably at least two hours, at a temperature of at least 95°C, preferably at least 98°C.
  • control unit is configured to control a process of cooling the water and feedstock after having heated the water and feedstock.
  • control unit is configured to control a process of adding a microorganism culture to the first reactor vessel.
  • process of adding a microorganism culture to the first reactor vessel is performed after having heated and cooled the water and feedstock.
  • control unit is configured to control a fermentation process in the first reactor vessel upon having added a microorganism culture to the first reactor vessel.
  • each of the first reactor vessel and the second reactor vessel have a volume of a least 200 liters.
  • the sensor for sensing a level of fermentation comprises a conductivity sensor and/or a brix meter and/or an optical sensor and/or a density and/or biomass sensor and/or sensor for sensing suspended solid.
  • control unit is configured to deactivate fermentation in the first reactor vessel when the seed train part of the first batch fermented feed has been transferred from the first reactor vessel to the second reactor vessel.
  • the predetermined level of fermentation is selected from a range of 55-90% of a final state of fermentation, preferably selected from a range of 60-80% of a final state of fermentation.
  • control unit is configured to control a process of transferring the seed train part of the first batch fermented feed from the first reactor vessel to the second reactor vessel and emptying a remaining production part of the first batch fermented feed from the first reactor vessel when the level of fermentation has reached the predetermined level of fermentation.
  • bioreactor system according to any one of items 20-21 , further comprising a mixer for mixing the production part of the first batch fermented feed with additional feed material, such as animal nutrition and/or acid, preferably further adapted for mixing a second batch fermented feed from the second reactor vessel with addition feed material.
  • additional feed material such as animal nutrition and/or acid
  • bioreactor system according to any one of the preceding items, further comprising one or more air inlets for supplying sterile air in the first reactor vessel and/or second reactor vessel.
  • bioreactor system according to any one of the preceding items, further comprising one or more members or devices for stirring, mixing and/or circulating content of the first reactor vessel and/or second reactor vessel.
  • control unit is configured to stop a process of adding water and start adding feedstock to the second reactor vessel when the water has reached a predefined water level.
  • control unit is configured to stop the process of adding feedstock to the second reactor vessel when the water and feedstock have reached a predefined water and feedstock level.
  • control unit is configured to control a fermentation process in the second reactor vessel upon having received the seed train part of the first batch fermented feed from the first reactor vessel.
  • control unit is configured to deactivate fermentation in the second reactor vessel and empty a second batch fermented feed from the second reactor vessel when the level of fermentation has reached a second predetermined level of fermentation.
  • the disinfection connection comprises a first membrane towards the first reactor vessel, a second membrane towards the microorganism vessel and a disinfection compartment disposed between the first membrane and the second membrane.
  • bioreactor system according to any one of the preceding items, further comprising at least one further third reactor vessel fluidly connected to the first reactor vessel and/or the second reactor vessel; one or more third vessel inlets for receiving feedstock and water; a third vessel fermented feed inlet for receiving fermented feed from the first and/or second reactor vessel; a third vessel outlet for removing third batch fermented feed from the third reactor vessel.
  • a method for production of fermented feed comprising: providing a bioreactor system comprising: a first reactor vessel comprising a first vessel outlet for removing first batch fermented feed from the first reactor vessel; a second reactor vessel fluidly connected to the first reactor vessel, the second reactor vessel comprising a vessel fermented feed inlet for receiving first batch fermented feed from the first reactor vessel; filling the first reactor vessel with water and feedstock; adding a microorganism culture to the first reactor vessel; controlling a fermentation process in the first reactor vessel; when a level of fermentation has reached a predetermined level of fermentation, transferring a seed train part of a first batch fermented feed from the first reactor vessel to the second reactor vessel through the vessel fermented feed inlet of the second reactor vessel; emptying a remaining production part of the first batch fermented feed from the first reactor vessel; controlling a fermentation process in the second reactor vessel; when a level of fermentation has reached a second predetermined level of fermentation, deactivating fermentation in the second reactor vessel; and emptying a

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Abstract

La présente divulgation concerne un système de bioréacteur pour la production agricole d'aliments fermentés. Le système comprend : une première cuve de réaction comprenant : un ou plusieurs orifices d'entrée de première cuve pour la réception d'une charge de départ, d'eau et d'une culture de microorganismes ; un orifice de sortie de première cuve pour l'évacuation d'une première charge fermentée par lots hors de la première cuve de réaction ; une seconde cuve de réaction en communication fluidique avec la première cuve de réaction ; un orifice d'entrée d'aliments fermentés de seconde cuve pour la réception d'aliments fermentés en provenance de la première cuve de réaction ; et une unité de commande servant à commander la production agricole d'aliments fermentés dans la première cuve de réaction et la seconde cuve de réaction. L'unité de commande est conçue pour : amener une vanne, située entre la première cuve de réaction et ledit ou lesdits orifices d'entrée d'aliments fermentés de seconde cuve de la seconde cuve de réaction, à transférer une partie de la chaîne d'ensemencement des premiers aliments fermentés par lots de la première cuve de réaction à la seconde cuve de réaction par l'intermédiaire de l'orifice d'entrée d'aliments fermentés de la seconde cuve lorsque les mesures en provenance d'un capteur servant à détecter un niveau de fermentation dans la première cuve de réaction indiquent qu'un processus de fermentation dans la première cuve de réaction a atteint un niveau prédéterminé de fermentation. La divulgation concerne en outre un procédé de production d'aliments fermentés.
PCT/EP2024/077443 2023-09-28 2024-09-30 Système de bioréacteur et procédé de production agricole d'aliments fermentés Pending WO2025068592A2 (fr)

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