Bioreactor system and methods

Abstract

A bioreactor system for microbial processing and related methods. The system includes a production reactor vessel for biomass fermentation; and a seed reactor vessel for developing seed inoculum for seeding fermentation in the production reactor vessel, the seed reactor vessel being fluidly connected to the production reactor vessel for sterile transferring of the seed inoculum thereto, the seed reactor vessel being configured to prepare the seed inoculum in parallel with operation of the production reactor vessel. A method for obtaining a microbial strain for use with the bioreactor system. The method includes culturing the microbial strain and fermenting the cultured microbial strain. A method for obtaining a microbial-based hydrolysate. The method including drying the microbial strain obtained from the bioreactor system, heating the strain to reduce RNA content, hydrolyzing the strain, filtering and centrifugating the microbial strain and pasteurizing the microbial strain to obtain a microbial-based hydrolysate.

Description

BIOREACTOR SYSTEM AND METHODSCROSS-REFERENCE

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 570,495, entitled “Liquid State Biomass Fermentation Bioreactor System,” filed on March 27, 2024, the entirety of which is incorporated by reference herein.FIELD OF TECHNOLOGY

[0002] The present technology relates to bioreactor systems and related methods.BACKGROUND

[0003] The advancement of protein science and technology has been significantly driven by the quest for sustainable and efficient methods of protein production. In this context, the utilization of lignocellulosic biomass from side-stream commodity crops presents a promising avenue. These biomass materials, often regarded as agricultural waste or byproducts, hold untapped potential for conversion into valuable protein products. However, the inherent complexity and recalcitrant nature of lignocellulosic materials pose considerable challenges, leading to several preprocessing steps, variations based on the final process, and varying degrees of metabolic and byproducts produced. Traditional methods for converting these materials into protein are often inefficient, environmentally burdensome, and economically unviable, primarily due to the need for intensive chemical or enzymatic treatments to break down the complex cellulose structures.

[0004] Recent developments have shifted focus towards biological conversion methods, utilizing microbes for the conversion of lignocellulosic and carbon-based materials into protein. This approach offers a greener and potentially more cost-effective alternative. However, the efficiency of microbial conversion and the quality of the resulting protein products are highly variable.

[0005] There therefore remains a need for systems and methods to address the above challenges.SUMMARY

[0006] It is an object of the present technology to ameliorate at least some of the inconveniences present in the prior art.

[0007] One of the critical challenges in utilizing lignocellulosic biomass is the preparation and processing of the raw materials. These materials, including beet pulp, canola meal, corn starch, and other agricultural byproducts, require specific treatments such as fractionation, pelletization, and preprocessing to become suitable substrates for microbial conversion. Additionally, the need for sterilization to prevent contamination and ensure a controlled conversion process further complicates the system.

[0008] The current invention aims to address at least some of these challenges by introducing a comprehensive system and process for the creation of mushroom mycelium from lignocellulosic side-stream commodity crops. This system encompasses a variety of innovative features. The system is capable of processing varied forms of material preparation (pellets, shreds, mash, etc.). Effective sterilization methods are available to maintain sterility of the system (steam sterilization under pressure, UV light sterilization methods). Advanced technologies can also be incorporated, such as Al computing, for process optimization. The system's design, made of stainless steel and acrylic or equivalent materials, ensures durability and compliance with industrial standards. Furthermore, the system integrates various external features such as water recycling and temperature control mechanisms, enhancing its sustainability and efficiency.

[0009] Notably, the system allows for the aseptic introduction of microbes and controlled titration methods, ensuring precision and consistency in the protein production process. The orientation of the system facilitates multiple fermentation tanks being fed through single or multiple preprocessing steps and expedited through single or multiple modular post-processing steps, optimizing the overall production flow.

[0010] According to one aspect of the present technology, there is provided a bioreactor system for microbial processing. The system includes a production reactor vessel for biomass fermentation; and a seed reactor vessel for developing seed inoculum for seeding fermentation in the production reactor vessel, the seed reactor vessel being fluidly connected to the production reactor vessel for sterile transferring of the seed inoculum thereto, the seed reactor vessel being configured and arranged to prepare the seed inoculum in parallel with operation of the production reactor vessel.

[0011] In some embodiments, the system further includes a first sterilization lamp arranged to sterilize the production reactor vessel; and a second sterilization lamp arranged to sterilize the seed reactor vessel.

[0012] In some embodiments, the first sterilization lamp extends into an interior of the production reactor vessel; and the first sterilization lamp is an ultraviolet-C light based sterilization lamp.

[0013] In some embodiments, the second sterilization lamp extends into an interior of the seed reactor vessel; and the second sterilization lamp is an ultraviolet-C light based sterilization lamp.

[0014] In some embodiments, the first sterilization lamp and the second sterilization lamp produce light centered at 254 nm.

[0015] In some embodiments, the production reactor vessel includes a vessel body having an opening, and a vessel lid sealingly closing the opening of the vessel body. The seed reactor vessel includes a seed vessel body having an opening, and a vessel lid sealingly closing the opening of the seed vessel body; the first sterilization lamp is connected to and supported by the vessel lid of the production reactor vessel; and the second sterilization lamp is connected to and supported by the vessel lid of the seed reactor vessel.

[0016] In some embodiments, the system further includes a water delivery assembly for providing input water to the seed reactor vessel and the production reactor vessel. The water delivery assembly includes a first water pump fluidly connected to at least one of the seed reactor vessel and the production reactor vessel, the first water pump being arranged to pump input water to the at least one of the vessels; and a second water pump fluidly connected to the seed reactor vessel and the production reactor vessel, the second water pump being arranged to pump wastewater out of the seed reactor vessel and the production reactor vessel.

[0017] In some embodiments, the water delivery assembly further includes at least one first flow-through water sterilizer in fluid communication with the first water pump for sterilizing the input water prior to introduction in the at least one of the vessels; and at least one second flow- through water sterilizer in fluid communication with the second water pump for sterilizing the wastewater expelled from the seed reactor vessel and the production reactor vessel.

[0018] In some embodiments, the system further includes at least one filter for filtering wastewater prior to sterilization.

[0019] In some embodiments, the at least one filter includes a submicron pore size filter.

[0020] In some embodiments, an aeration system operatively connected to the production reactor vessel. The aeration system includes a sparger disposed in the production reactor vessel; and an air pump fluidly connected to the sparger, during operation, the air pump causing air to flow into the production reactor vessel through the sparger to aerate contents in the production reactor vessel.

[0021] In some embodiments, the air pump is further fluidly connected to the seed reactor vessel for aerating contents in the seed reactor vessel during operation thereof.

[0022] In some embodiments, the system further includes a compressor system fluidly connected to the seed reactor vessel and the production reactor vessel for collecting humidity from air expelled from the vessels.

[0023] In some embodiments, the system further includes an agitator disposed in the production reactor vessel for agitating contents of the production reactor vessel during operation.

[0024] In some embodiments, the agitator includes an impeller disposed in an interior of the production reactor vessel; and a motor disposed on an exterior of the production reactor vessel, the impeller being magnetically coupled to the motor for driving thereby.

[0025] In some embodiments, the system further includes a plurality of sensors connected to the production reactor vessel, the plurality of sensors being arranged and configured to sense operating conditions in the production reactor vessel.

[0026] In some embodiments, the plurality of sensors include at least one of a pH sensor; a dissolved oxygen (DO) sensor; a temperature sensor; and a camera.

[0027] In some embodiments, the production reactor vessel includes a vessel body having an opening, and a vessel lid sealingly closing the opening of the vessel body; and at least one of the plurality of sensors is connected to and supported by the vessel lid.

[0028] In some embodiments, the system further includes a computer-implemented system operatively connected to the plurality of sensors, the computer-implemented system including a human-machine interface (HMI) for providing information to an operator and receiving commands from the operator for controlling operation of the bioreactor system.

[0029] In some embodiments, the system further includes at least one peristaltic pump fluidly connected between the seed reactor vessel and the production reactor vessel for pumping the seed inoculum to the production reactor vessel.

[0030] In some embodiments, the system further includes a pH control system operatively connected to the production reactor vessel for managing pH levels of fermentation during operation.

[0031] According to another aspect of the present technology, there is provided a method for obtaining a microbial strain for use with a bioreactor system. The method includes: i) culturing the microbial strain on a culture agar medium in presence of yeast extract, glucose, and peptone for a culture period of between about 18 hours and about 144 hours; and ii) fermenting the cultured microbial stain of step i) on a fermentation agar medium in presence of yeast extract, glucose, and peptone for a fermentation period of between about 18 hours and about 144 hours.

[0032] In some embodiments, step i) is performed at a pH of between about 5.5 and about 7.5.

[0033] In some embodiments, step ii) is performed at a pH of between about 5.5 and about 7.5.

[0034] In some embodiments, step i) is performed at a temperature of between about 20°C and about 30°C.

[0035] In some embodiments, step ii) is performed at a temperature of between about 20°C and about 30°C.

[0036] In some embodiments, the microbial strain is a fungal strain.

[0037] In some embodiments, the microbial strain is a mycelium.

[0038] According to another aspect of the present technology, there is provided a method for obtaining a microbial-based hydrolysate. The method includes: i) drying the microbial strain obtained from a bioreactor system; ii) heating the dried microbial strain of step i) to reduce RNA content;iii) hydrolyzing the microbial strain of step ii); iv) filtering and centrifugating the microbial strain of step iii); and vi) pasteurizing the microbial strain of step iv) to obtain a microbial -based hydrolysate.

[0039] In some embodiments, prior to step i), the microbial strain is rinsed with water at a temperature of between 10°C about and about 30°C.

[0040] In some embodiments, step ii) is carried out at a temperature of about 60°C for about 1 hour.

[0041] In some embodiments, step ii) is carried out using microwave.

[0042] In some embodiments, step iii) is carried out using acid, alkaline pH, heat, enzymes or fermentation.

[0043] In some embodiments, step iii) is carried out using proteases.

[0044] In some embodiments, the microbial strain is a fungal strain.

[0045] In some embodiments, the microbial strain is a mycelium.

[0046] For purposes of this application, terms related to spatial orientation such as top, bottom, left, and right, are used for simplicity of reference. It is understood that some arrangements may rely on gravitational forces to operate, although generally the spatial orientation simply provides an arrangement of components relative to one another.

[0047] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. Like numbers refer to like elements throughout. As used herein, phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y. As used herein, phrases such as “between about X and Y” mean “between about X and about Y.” As used herein, phrases such as “from about X to Y” mean “from about X to about Y.”

[0048] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaningin the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity and / or clarity.

[0049] The term “automatically” means that the operation can be substantially, and typically entirely, carried out without human or manual input, and is typically programmatically directed or carried out. The term “electronically connected” includes both wireless and wired connections between components. The term “about” means that the recited parameter or value can vary by between about + / -20%.

[0050] Embodiments of the present technology each have at least one of the above-mentioned objects and / or aspects, but do not necessarily have all of them. It should be understood that some aspects of the present technology that have resulted from attempting to attain the above- mentioned object may not satisfy this object and / or may satisfy other objects not specifically recited herein.

[0051] Explanations and / or definitions of terms provided in the present application take precedence over explanations and / or definitions of these terms that may be found in any documents incorporated herein by reference.

[0052] Additional and / or alternative features, aspects and advantages of embodiments of the present technology will become apparent from the following description, the accompanying drawings and the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0053] For a better understanding of the present technology, as well as other aspects and further features thereof, reference is made to the following description which is to be used in conjunction with the accompanying drawings, where:

[0054] Figure 1 is a perspective view of a bioreactor system according to non-limiting embodiments of the present technology;

[0055] Figure 2 is a perspective view of the bioreactor system of Figure 1 , with a housing removed;

[0056] Figure 3 is a front elevation view of the bioreactor system of Figure 1 ;

[0057] Figures 4 and 5 are side elevation views of the bioreactor system of Figure 1 ;

[0058] Figure 6 is a schematic diagram of the bioreactor system of Figure 1 ;

[0059] Figure 7 is a top view of a lid of a production reactor vessel of the bioreactor system ofFigure 1 ;

[0060] Figure 8 is a top view of a lid of a seed reactor vessel of the bioreactor system of Figure 1 ;

[0061] Figure 9 is a flowchart illustrating a method for obtaining a microbial strain for use with the bioreactor system of Figure 1 ;

[0062] Figure 10 is a flowchart illustrating a method for obtaining a microbial-based hydrolysate following operation of the bioreactor system of Figure 1 ; and

[0063] Figure 11 is a schematic chart of steps to producing a microbial-based hydrolysate using the bioreactor system of Figure 1.

[0064] It should be noted that the Figures may not be drawn to scale.DETAILED DESCRIPTION

[0065] The present technology is described below with respect to the production of mycelium and mushroom protein through biomass fermentation. It is contemplated that the bioreactor system could be employed for biomass fermentation for the production of different resultant products.

[0066] The liquid state biomass fermentation bioreactor system 100 described herein is designed to operate in parallel, enhancing efficiency and scalability in the production process. By the present embodiment, the system 100 is designed for the creation of mushroom mycelium from lignocellulosic side-stream commodity crops. The system 100 utilizes microbial processes to convert lignocellulosic carbon and nitrogen-based material into protein. It is designed to handle a variety of materials derived from agricultural byproducts including but not limited to: beet pulp, canola meal, pea starch, corn starch, distillers grain, compost, dextrose, pea protein, and / or a mixture thereof. These materials can be presented in various forms including pellets, shreds, mash, pulp, or particles.

[0067] In the present embodiment, the microbial I fungal strain chosen is the Pleurotus ostreatus strain, which has a relatively strong lignocellulosic material degradation capability.

[0068] The selected strain, also known as the Oyster Mushroom strain, further aids in maximizing the use of growing space. This strain generally outperforms other strains in mycoprotein production, thereby producing more mycoprotein per volume of production. The efficiency of its semi-continuous liquid state fermentation is notably higher than that of other methods like gas fermentation.

[0069] Use of this microbial strain further permits the system to operate at ambient temperature. This in turn generally reduces the need for external energy inputs, e.g. to warm or cool the system.

[0070] Mycelium, the vegetative part of a fungus, and mushroom protein are increasingly recognized for their nutritional and environmental benefits. They are a sustainable source of protein and other nutrients, with a wide range of applications in food, pharmaceuticals, and other industries. Traditional methods of mycelium and mushroom cultivation often face limitations in scalability, contamination control, and efficiency. The present system 100 is an approach to overcome at least some of these obstacles.

[0071] With reference to Figures 1 to 6, a bioreactor system 100 for microbial processing is illustrated according to a non-limiting embodiment of the present technology.

[0072] As is illustrated in Figures 1 and 3, the system 100 includes a housing 102 in which the components of the system 100 are disposed. The particular shape, form, and materials of the housing 102 could vary in different embodiments and are not meant to be particularly limited. In the illustrated embodiment, the housing 102 is formed from a stainless steel frame and acrylic windows, to provide structural strength while also permitting visual inspection of the fermentation occurring in the system 100 during operation. As can be seen in Figure 2, most components of the system 100 in the present embodiment are mounted on translating rails, such that the components can be easily accessed if maintenance is desired or required.

[0073] The system 100 includes two production reactor vessels 110 for biomass fermentation, also referred to herein as the vessel 110. The production reactor vessels 110 are arranged and configured to support the liquid state biomass fermentation of the system 100. The productionreactor vessels 110 in the present embodiment are identical and as such only one production reactor vessel 110 will be described in detail.

[0074] The vessel 110 includes a vessel body 112 having at least one opening at one end of the vessel body 112. In the present embodiment, the vessel body 112 is formed from a cylindrical body with open ends. A vessel lid 114 sealingly closes a top opening of the vessel body 112. A similar lid 116, referred to herein as an end cap 116, sealingly closes a bottom opening of the vessel body 112. The lid 114 is selectively connected to the vessel body 112, such that it may be removed for maintenance inside the vessel body 112. The lid 114 and the end cap 116 have apertures defined therein to permit the flow of material, water, air, etc into and out of the vessel 110 (described in more detail below).

[0075] The production reactor vessel 110 includes an agitator 120 (schematically illustrated in Figures 5 and 6) operatively connected thereto for agitating contents of the production reactor vessel 110 during operation. Agitation ensures uniform distribution of nutrients and microbes throughout the biomass, enhancing the fermentation process.

[0076] By the present embodiment, the agitator 120 includes an impeller 122 disposed in an interior of the production reactor vessel 110. The agitator 120 further includes a motor 124 disposed on an exterior of the vessel 110. The particular positioning of the motor 124 and other components of the agitator 120 may vary in different embodiments. As one non-limiting example, the motor 124 and control components related thereto could be connected to and / or supported by the end cap 116.

[0077] The impeller 122 is magnetically coupled to the motor 124. The motor 124 thus provides power and rotation to the impeller 122 inside the vessel 110, while avoiding components such as a drive shaft inside the vessel 110 that could require additional maintenance or cleaning.

[0078] The system 100 also includes two seed reactor vessels 130, also referred as the vessels 130. Each seed reactor vessel 130 is operatively connected to a corresponding one of the production reactor vessels 110 for developing seed inoculum for seeding fermentation for operation of the corresponding production reactor vessel 110. Each seed reactor vessel 130 is fluidly connected to the corresponding production reactor vessel 110 for sterile transferring of the seed inoculum thereto. The seed reactor vessels 130 in the present embodiment are identical and as such only one seed reactor vessel 130 will be described in detail.

[0079] The vessel 130 includes a vessel body 132 having at least one opening at one end of the vessel body 132. In the present embodiment, the vessel body 132 is formed from a cylindrical body with open ends. A vessel lid 134 sealingly closes a top opening of the vessel body 132. A similar lid 136, referred to herein as an end cap 136, sealingly closes a bottom opening of the vessel body 132. The lid 134 is selectively connected to the vessel body 132, such that it may be removed for maintenance inside the vessel body 132. The lid 134 and the end cap 136 have apertures defined therein to permit the flow of material, water, air, etc into and out of the vessel 130 (described in more detail below).

[0080] The seed reactor vessel 130, also referred to as the seed bioreactor 130, is configured and arranged to prepare seed inoculum in parallel with operation of the production reactor vessel 110. By having a separate seed-developing vessel 130, a seeding stage of the fermentation can be commenced, in the seed reactor vessel 130, while a previous cycle of fermentation is still in process in the production reactor vessel 110.

[0081] The seed bioreactor 130 thus enables significant production capacity increase and shortened timescales as it enables parallelized growth. The seed bioreactor 130 is inoculated with dried, sterile mycelium in the present embodiment to ensure sterility. The inoculum will be ready for use in the production reactor vessel 110 within about one week; during this week, the vessel 110 can be in production.

[0082] The seed reactor vessel 130 is fluidly connected to the production reactor vessel 110 in order to perform a sterile transfer of the seed inoculum from the vessel 130 to the vessel 110. Silicone tubing connects the bottom end cap 136 of the vessel 130 to the lid 114 of the production reactor vessel 110 for a sterile transfer of the cultured seed inoculum. One or more pumps 160 are fluidly connected to the tubing to drive the transfer therethrough. In the present embodiment, one or more peristatic pumps 160 are used for the transfer of the seed inoculum from the vessel 130 to the vessel 110.

[0083] In at least some implementations or operations of the system 100, a small percentage of the culture prepared in the seed bioreactor 130 could be left in the vessel 130 to be used as the inoculum for a next seed batch.

[0084] The vessels 110, 130 generally have a similar design arrangement, however the seed vessel 130 is smaller. In the illustrated embodiment, the production reactor vessel 110 has a volume of about 25 liters and the seed reactor vessel 130 has a volume of about 2 liters.

[0085] The exact volumes and the ratio of volumes between the vessels 110, 130 are not meant to be limited to the illustrated example. The seed reactor vessel 130 is smaller than the production reactor vessel 110, both as the required volume is less and to reduce the overall footprint of the system 100.

[0086] As is illustrated in Figures 1 to 5, the system 100 could include additional pairs of the production vessel 110 and the seed vessel 130. One seed reactor vessel 130 would be included and paired with each corresponding production reactor vessel 110. By including additional pairs of vessels 130, the total production of the system 100 can be increased. Using cylindrical vessels 110, 130, the pairs of vessels 110, 130 can be arranged in parallel inside the housing 102.

[0087] With additional reference to Figures 7 and 8, the connections provided in the present embodiment by the lids 114, 134 and the end caps 116, 136 are described in additional detail.

[0088] The lid 114 of the production reactor vessel 110 has various apertures formed therein to connect an interior of the vessel 110 to components or assemblies of the system 100. The lid 134 of the seed reactor vessel 130 similarly has various apertures formed therein to connect an interior of the vessel 130 to components or assemblies of the system 100.

[0089] The end caps 116, 136 of the vessels 110, 130 also provide points of connection (not shown) which permit connections between the interiors of the vessels 110, 130 and components exterior thereto.

[0090] Using the connections with the lids 114, 134 and the end caps 116, 136, water, media, air, and inoculum can be moved into or out of the vessels 110, 130 automatically and / or without user manipulation. This arrangement aids in maintaining sterility of the system 100 and aids in reducing contamination.

[0091] The lid 114 of the vessel 110 has a pair of UV light apertures 90. The vessel 110 includes two sterilization lamps 119 arranged to sterilize an interior of the production reactor vessel 110. In some embodiments, the vessel 110 could include more or fewer lamps 119.

[0092] The sterilization lamps 119 extend into an interior of the production reactor vessel 110 through the lid 114. The lid 114 supports the lamps 119, with upper ends portions of the lamps 119 being connected to the lid 114. The lamps 119 are thus suspended inside the vessel body

[0093] The lamps 119 are configured and arranged to sterilize interior surfaces of the vessel 110 by ultraviolet radiation. The sterilization lamp 119 for the present application is specifically a pulsed, high-intensity ultraviolet-C light sterilization lamp 119. It is contemplated that for other applications, the specific waveband or type of lamp may vary.

[0094] The lid 134 of the vessel 130 has one LIV light aperture 80 defined therein. The vessel 130 includes a sterilization lamp 139 arranged to sterilize an interior of the seed reactor vessel 130. In some embodiments, the vessel 130 could include additional lamps 139.

[0095] The sterilization lamp 139 extends into an interior of the seed reactor vessel 130 through the lid 134. The lid 134 supports the lamp 139 by an upper end portion thereof. The lamp 139 is thus suspended inside the vessel body 132.

[0096] The lamp 139 is configured and arranged to sterilize interior surfaces of the vessel 130 by ultraviolet radiation. The sterilization lamp 139 for the present application is specifically a pulsed, high-intensity ultraviolet-C light sterilization lamp 139.

[0097] In the present embodiment, the sterilization lamps 119, 139 produce light centered at 254 nm. The precise center wavelength and bandwidth of the lamps 119, 139 may vary in different embodiments.

[0098] The LIV-C light band (200-280 nm, especially 253.7 nanometers) can effectively kill yeast, molds, bacteria, viruses, and algae. For the present embodiment, the LIV-C light band is employed for both the relative energy efficiency (compared to other sterilization methods) and its ability to disinfect both gasses and liquids. Several LIV bulbs are implemented throughout the process as double checks. Other potential benefits of LIV-C as a primary disinfection method include high reliability, infrequent maintenance, and scalability.

[0099] It is contemplated that for other applications, the specific waveband or type of lamp may vary. It is also contemplated that the lamps 119 and the lamp 139 could be chosen differently to address different disinfection needs of the two vessels 110, 130 in some embodiments.

[0100] The lamps 119, 139, as well as the LIV water sterilization (described below), are included to maintain sterility in the system 100. UVA-LED methods of sterilization have been found to inactivate bacteria, such as Escherichia coli DH5a, Enteropathogenic E. coli, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella enterica serovar Enteritidis. In combination with decontaminants such as hydrogen peroxide (35%), and reverse osmosisfiltration, a broad spectrum of pathogen control has been successfully implemented throughout the system 100. A 3- to 6-log reduction in the influent microbial levels occurs following LIV disinfection. Absorption of the adequate LIV dosage by the microorganisms at the 254 nm wavelength results in a greater than 99.9% reduction in the influent microbial population. Most of the common waterborne pathogenic microorganisms can be inactivated using a LIV dosage of less than 15,000 pW-sec / cm2 and at 253.7 nm and 26,400 pW-sec / cm2, 100% of the most resistant bacteria are deactivated. A minimum of greater than 4-log reduction is required for the influent viral population.

[0101] As is noted above, sterilization of the vessel 110 could include the use of cleaning agents or decontaminants, such as hydrogen peroxide (35%), to assist with cleaning the vessel 110 before and / or after operation of the system 110. The lid 114 has defined therein two nozzle apertures 91 for connecting nozzles (not shown) for delivering decontaminants to an interior of the vessel 110. It is contemplated that the lid 134 of the seed reactor vessel 130 could have one or more nozzle apertures defined therein in some embodiments. It is also contemplated that one or more nozzles could be operatively connected to a steam system, to permit steam disinfection of the vessel 110 and / or the vessel 130, alternatively or in addition to the above mentioned disinfection arrangements.

[0102] The lid 114 of the vessel 110 has one air outlet aperture 92 defined therein to permit gas expelled from the vessel 110 to be directed out of the system 100. The lid 134 of the vessel 130 also has one air outlet aperture 82 defined therein to permit gases expelled from the vessel 130 to be directed out of the system 100. In the present embodiment, tubing (shown schematically in Fig. 6) is connected to each of the apertures 82, 92 to direct the gases out of the housing 102.

[0103] In the present embodiment, the system 100 further includes a condenser 149 fluidly connected to the air outlet apertures 82, 92. The condenser 149 removes at least some humidity from the expelled gases.

[0104] In at least some embodiments, the water extracted by the condenser 149 from the expelled gases could be re-used in the system 100. In such an embodiment, the system 100 could be nearly fully contained, thereby conserving water and reusing of resources, aligning with controlled environment agriculture technologies.

[0105] The lid 134 of the vessel 130 further has one water inlet aperture 84 defined therein to permit clean input water to be added to the vessel 130 from a water delivery assembly 170(described in detail below). In the present embodiment, input water from the water delivery assembly 170 is delivered to the vessel 110 through the end cap 116 (not shown). It is contemplated that an aperture for input water could be defined in the lid 114 in some embodiments.

[0106] As is illustrated schematically in Figure 6, the system 100 includes a pH control system 180 operatively connected to the production reactor vessel 110 for managing pH levels of fermentation during operation. The pH control system 180 includes a pH sensor 181 operatively connected to the vessel 110 and communicatively connected to a controller 99 of the system 100 (described below). The lid 114 of the vessel 110 further has a pair of pH control apertures 93 defined therein. One aperture 93 is operatively connected to an acid source; the other aperture 93 is operatively connected to a base source of the pH control system 180. In response to the detected pH and according to control methods of the controller 99, the pH control system 180 can cause delivery of either an acid or base in order to manage the pH levels in the production reactor vessel 110 during operation of the system 100.

[0107] The lid 134 of the vessel 130 further has one inoculum inlet aperture 86 defined therein to permit input inoculum to be added to the vessel 130.

[0108] To begin preparing the seed inoculum in the seed reactor vessel 130, preserved microbial cultures are introduced into the vessel 130 through the aperture 86. In the present embodiment, these are long-term preserved microbial cultures, stored in sterile distilled water for 3-30 yrs at 5° C. Due to the absence of the nutrients, the metabolic activity is hampered, and the microbe attains a dormant state. When the culture is provided with nutrients, the activity is reviewed. The longer shelf life of the inoculum is advantageous during prolonged field or space missions, for example.

[0109] The system 100 includes fixed mounts (not shown) for an inoculum reservoir (not shown) containing the preserved microbial cultures. In some embodiments, the inoculum reservoir is specifically an inoculum preservation bottle which is designed to be periodically replaced. One or more bottles are connected via quick-connect aseptic fittings, allowing for straightforward replacement and refilling without compromising sterility.

[0110] In some embodiments, a user manages the introduction of the dormant cultures into the vessel 130 through the aperture 86 manually aseptically. It is also contemplated that thesystem 100 could include a culture reservoir fluidly connected to the vessel 130 and that the dormant cultures could be pumped into the vessel 130 automatically.

[0111] The lid 134 of the vessel 130 further has one media inlet aperture 88 defined therein to permit input nutrient media to be added to the vessel 130. In order to activate and begin cultivating the microbes (mycelium in the present embodiment), a small amount of the nutrient media is added to the vessel 130.

[0112] In the illustrated embodiment, the system 100 includes a media reservoir 105 fluidly connected to the vessel 130 via a peristaltic pump 107 and the aperture 88. In some embodiments, a user could manage the introduction of the media into the vessel 130 through the aperture 88 manually.

[0113] The system 100 includes fixed mounts (not shown) for the media reservoir 105. In some embodiments, the media reservoir 105 is specifically a media concentrate bottle 105 which is designed to be periodically replaced. One or more bottles are connected via quick-connect aseptic fittings, allowing for straightforward replacement and refilling without compromising sterility. Inoculum sources follow a similar approach, enabling external preparation and easy integration.

[0114] The system 100 is configured and arranged to convert lignocellulosic carbon and nitrogen-based material ( / .e. media) into protein using microbial processes. The system 100 and methods presented herein allow conversion of a variety of materials derived from agricultural byproducts, including but not limited to beet pulp, canola meal, pea starch, corn starch, distillers’ grain, compost, dextrose, pea protein, or a mixture thereof. These materials can be presented in various forms including pellets, shreds, mash, pulp, or particles. Processing and sterilization of the media is performed prior to introduction into the system 100.

[0115] The lid 114 of the vessel 110 further has one inoculum inlet aperture 94 defined therein to permit input the seed inoculum to be added to the vessel 110 from the seed vessel 130. The tubing and pump 160 fluidly connect to an interior of the vessel 110 via the aperture 94 to provide automatic and sterile transfer of the seed inoculum after preparation in the seed vessel 130.

[0116] The lid 114 of the vessel 110 further has one media inlet aperture 95 defined therein to permit input nutrient media to be added to the vessel 110.

[0117] In the illustrated embodiment, the media is delivered to the vessel 110 from the media reservoir 105, also fluidly connected to the vessel 110, by the peristaltic pump 107 via the aperture 95.

[0118] The pump 107 enables flow control so that the media, previously sterilized, can be moved to the vessels 110, 130 without contamination. Peristaltic pumps have been chosen in the present case as they allow for careful flow control and are in a closed system, aiding in maintaining sterility.

[0119] The media reservoir 105 of the present embodiment is an autoclavable, LIV resistant 2L bottle, with modified lid for fluidly connecting to the pump 107.

[0120] In some embodiments, a user could manage the introduction of the media into the vessel 110 through the aperture 95 manually.

[0121] With continued reference to Figure 6, the system 100 further includes an aeration system 140 for providing aeration and oxygenation to the production reactor vessel 110 during operation. In the present embodiment, the aeration system 140 also provides aeration and oxygenation to the seed reactor vessel 130 during operation. Proper ventilation (aeration) is critical for maintaining the desired gas composition, which is essential for the growth and development of mycelium.

[0122] The aeration system 140 includes an air pump 141 fluidly connected to the production reactor vessel 110 and the seed reactor vessel 130 for aerating the fluids therein during operation. In some embodiments, separate air pumps could be provided for the vessels 110, 130.

[0123] The aeration system 140 includes a sparger 142 disposed in an interior of the production reactor vessel 110, connected to the air pump 141 by tubing. The sparger 142 aids in controlling bubble size. The particular bubble size aids in optimizing oxygen mass transfer into the liquid phase, ensuring efficient gas exchange within the production reactor vessel 110.

[0124] The aeration system 140 also includes a sparger 144 disposed in an interior of the seed reactor vessel 130, connected to the air pump 141 by tubing. The sparger 144 aids in controlling bubble size during seed inoculum preparation.

[0125] The air pump 141 causes air to flow into the vessels 110, 130 through the spargers 142, 144 to aerate contents in the vessels 110, 130.

[0126] The aeration system 140 also includes a filter 145 to remove physical contaminants from air prior to entry into the vessels 110, 130 through the spargers 142, 144. In the present embodiment, the filter 145 is a 0.2 pm pore size filter 145.

[0127] With continued reference to Figure 6, the system 100 further includes a water delivery assembly 170 for providing input water to the seed reactor vessel 130 and the production reactor vessel 110.

[0128] The water delivery assembly 170 includes one or more input water pumps 172 fluidly connected to the seed reactor vessel 130 and the production reactor vessel 110. One input pump 172 is schematically illustrated, but it is contemplated that each vessel 110, 130 could be provided with a corresponding pump 170.

[0129] The water delivery assembly 170 also includes various solenoid valves 174 for controlling water introduction into each of the vessels 110, 130. The solenoid valves 174 provide a relatively inexpensive solution to automation of valve control. Many such valves have an anti- water-hammer feature, avoiding sudden pressure differences caused by sudden valve closure. As such, use of solenoid valves may assist in extending the life of upstream components. The particular connection arrangement for a given valve 174 is chosen for both set-up convenience and maintenance.

[0130] The water pump 172 and the valves 174 fluidly connect a water input source to the vessels 110, 130 to provide clean input water thereto. In the present embodiment, the system 100 can be fluidly connected to an external water source. In some embodiments, it is contemplated that the system 100 could be provided with a clean water reservoir, either inside the housing 102 or external to the housing 102.

[0131] Input water is pumped into the seed reactor vessel 130 through the aperture 84 of the lid 134 thereof as noted above. In the illustrated embodiment, input water is pumped into the production reactor vessel 110 through the end cap 114 thereof, although the particular placement of the water input may vary.

[0132] The water delivery assembly 170 also includes one or more output water pumps 176 fluidly connected to the seed reactor vessel 130 and the production reactor vessel 110. One pump 176 is illustrated for the present embodiment, but it is contemplated that each vessel 110, 130 could be provided with a corresponding output pump. The output water pump 176 is arranged topump wastewater out of the seed reactor vessel 130 and the production reactor vessel 110 after operation thereof. It is also contemplated that the pump 176 could be used to evacuate cleaning agents introduced to the production reactor vessel 110.

[0133] In order to maintain sterility in the system 100 and to aid in maintaining safety of the system 100, the water delivery assembly 170 further includes water sanitizers for sterilizing input water flowing into the vessels 110, 130 and wastewater flowing out of the vessels 110, 130.

[0134] One flow-through water sterilizer 173 is disposed in fluid communication with the input water pump 172. The sterilizer 173 is configured and arranged to sterilize the input water prior to introduction in the vessels 110, 130. In the present embodiment, the sterilizer 173 is a UV-based sterilization lamp through which water is flowed. It is contemplated that different water sterilization arrangements could be employed.

[0135] Another flow-through water sterilizer 177 is disposed in fluid communication with the wastewater pump 176. The sterilizer 177 is configured and arranged to sterilize the wastewater received from the vessels 110, 130 and prior to re-se (either within the system 100 or outside the system 100). In the present embodiment, the sterilizer 177 is a UV-based sterilization lamp through which water is flowed. It is contemplated that different water sterilization arrangements could be employed.

[0136] The assembly 170 also includes one or more filters 178 (shown schematically) for filtering wastewater prior to sterilization. The filter 178 removes particulates from the wastewater. In addition to physically removing contaminants from the wastewater, the filter 178 aids in improving the UV sterilization efficiency as the presence of additional material in the water can decrease sterilization efficiency. In at least some embodiments, the one or more filters 178 include a submicron pore size filter, in some cases more specifically a 0.22 pm pore size filter.

[0137] With continued reference to Figure 6, the system 100 further includes a plurality of sensors (shown schematically) connected to the production reactor vessel 110. The sensors are arranged and configured to sense operating conditions in the production reactor vessel 110.

[0138] In the present embodiment, the sensors include the pH sensor 181 mentioned above, a dissolved oxygen (DO) sensor 190, a temperature sensor 192, and a camera 194. The camera 194 can provide visual monitoring to an operator and / or allow for image processing-based automatic monitoring by a computer. The sensors could be connected in a variety of positions onor in the vessel 110. In at least some embodiments, one or more of the sensors could connected to and supported by the vessel lid 114.

[0139] In order to manage operation of the bioreactor system 100, there is included a computer-implemented system 98, also referred to as a controller 98 or computer 98. The controller 98 is operatively and communicatively connected to the sensors 181 , 190, 192, 194. The sensors and controller 98 are configured to continuously monitor critical parameters such as pH, oxygen levels, digestible carbon, and protein levels. This real-time monitoring enables precise control over the fermentation process, ensuring optimal growth conditions and high-quality end products. In at least some embodiments, the controller 98 could be connected to an external computer-implemented system or device for remote and / or Al-optimized environmental control.

[0140] The controller 98 includes a human-machine interface (HMI) 99 for providing information to an operator and for receiving commands from the operator for controlling operation of the bioreactor system 100. The HMI 99 could be implemented in a variety of ways, including for instance through a touch-screen and / or input devices (keyboard, mouse).

[0141] The bioreactor system 100 described above may be used in combination with a method 200 for obtaining a microbial strain for use in the fermentation process mentioned above; see Figure 9. The method 200 includes selecting fungal strains that produce high protein biomass and storing the selected fungal strains.

[0142] The method 200 begins, at step 210, with culturing the microbial strain on a culture agar medium in presence of yeast extract, glucose, and peptone for a culture period of between about 18 hours and about 144 hours.

[0143] In some implementations, the culturing of step 210 is performed at a pH of between about 5.5 and about 7.5.

[0144] In some implementations, the culturing of step 210 is performed at a temperature of between about 20°C and about 30°C.

[0145] The method 200 continues, at step 220, with fermenting the cultured microbial stain of step 210 on a fermentation agar medium in presence of yeast extract, glucose, and peptone for a fermentation period of between about 18 hours and about 144 hours.

[0146] In some implementations, the fermenting of step 220 is performed at a pH of between about 5.5 and about 7.5.

[0147] In some implementations, the fermenting of step 220 is performed at a temperature of between about 20°C and about 30°C.

[0148] In some implementations of the method 200, the microbial strain is a fungal strain.

[0149] In some implementations of the method 200, the microbial strain is a mycelium.

[0150] As one non-limiting example, the method 200 includes culturing on a YPG or PDA agar medium including 10 g / L of yeast extract, 20 g / L of peptone and 20 g / L of glucose at 20 to 30° C., pH of 5.5 to 7.5, within the range of 18 to 36 or 72, or 96, or 120, or 144 hours, to refine each strain. The method 200 then continues with fermenting the cultured microbial strain on the YPG or PDA agar medium including 10 g / L of yeast extract, 20 g / L of peptone and 20 g / L of glucose at 20 to 30° C, pH of 5.5 to 7.5, between 18 to 36 or 72, or 96, or 120, or 144 hours.

[0151] Once the conversion process is complete, the bioreactor system 100 is configured and arranged to expel the fermented material from the production reactor vessel 110 onto conveyors (not shown) for further downstream processing. In the present embodiment, processed material is expelled by opening a solenoid valve (not shown) fluidly connected to the production reactor vessel 110. If the system 100 is operating in a gravity-enabled environment, gravity alone may facilitate the discharge. In microgravity or where additional force is needed, internal pressure is increased via controlled aeration by the aeration system 140, allowing the processed material to be expelled without the need for mechanical pumps.

[0152] The processing, in some implementations, includes centrifugation and dewatering. This feature facilitates the efficient transition from the conversion process to the final product preparation.

[0153] Following use of the bioreactor system 100, a method 300 can be used for obtaining a microbial-based hydrolysate from mycelium protein produced by the bioreactor system 100. A flowchart representing the method 300 is illustrated in Figure 10; additional method information is illustrated in the chart of Figure 11.

[0154] While the method 300 is described herein for obtaining the microbial-based hydrolysate from mycelium protein, it is contemplated that the method 300 could be applied to other microbialstrains. In some implementations, the method 300 could be applied to a microbial strain which is a fungal strain

[0155] A hydrolysate in this context is a product derived from the hydrolysis of mycelium protein, which is obtained through submerged fermentation in the bioreactor system 100. The hydrolysis process described in method 300 involves breaking down the complex protein structures of mycelium into smaller, more digestible fragments such as peptides and amino acids. The hydrolysis converts these proteins into a form that is easier for organisms to assimilate, enhancing their nutritional and functional properties.

[0156] The hydrolysates contain peptides, amino acids, minerals, carbohydrates, and lipids. One proposed use of the mycelium-based hydrolysate is as a supplement in the media of cellular agriculture as a means to reduce costs and increase the success of culturing due to adding all necessary amino acids and other components such as Beta-glucans.

[0157] The method 300 begins, at step 310, with drying mycelium protein material produced by the bioreactor system 100.

[0158] In some implementations, the mycelium protein material could be rinsed with water at a temperature of between 10°C about and about 30°C prior to step 310.

[0159] The method 300 continues, at step 320, with heating the dried microbial strain of step 310 to reduce RNA content.

[0160] Mycelium protein is dried at 60 degrees Celsius for RNA reduction for 1 hour. Following the temperature is reduced to between 10 and 50 degrees Celsius for example 10 degrees, 15 degrees, 20 degrees, 25 degrees, 30 degrees, 35 degrees, 40 degrees, 45 degrees, and 50 degrees to complete the drying process.

[0161] In some embodiments, the mycelium is heated in a water bath at 60 degrees Celsius for RNA reduction for 1 hour.

[0162] In some embodiments, the mycelium is microwaved for RNA reduction.

[0163] The method 300 continues, at step 330, with hydrolyzing the mycelium after RNA reduction.

[0164] It is contemplated that hydrolyzing the mycelium in step 330 could be carried out using acid, alkaline pH, heat, enzymes, or fermentation.

[0165] It is also contemplated that hydrolyzing the mycelium in step 330 could be carried out using proteases.

[0166] The method 300 then continues, at step 340, with filtering and centrifugating the hydrolyzed mycelium.

[0167] The method 300 then completes, at step 350, with pasteurizing the material of step 340 to obtain the microbial- or mycelium-based hydrolysate.

[0168] Modifications and improvements to the above-described implementations of the present technology may become apparent to those skilled in the art. The foregoing description is intended to be exemplary rather than limiting. The scope of the present technology is therefore intended to be limited solely by the scope of the appended claims.

Claims

What is claimed is:1 . A bioreactor system for microbial processing, the system comprising: a production reactor vessel for biomass fermentation; and a seed reactor vessel for developing seed inoculum for seeding fermentation in the production reactor vessel, the seed reactor vessel being fluidly connected to the production reactor vessel for sterile transferring of the seed inoculum thereto, the seed reactor vessel being configured and arranged to prepare the seed inoculum in parallel with operation of the production reactor vessel.

2. The system of claim 1 , further comprising: a first sterilization lamp arranged to sterilize the production reactor vessel; and a second sterilization lamp arranged to sterilize the seed reactor vessel.

3. The system of claim 2, wherein: the first sterilization lamp extends into an interior of the production reactor vessel; and the first sterilization lamp is an ultraviolet-C light based sterilization lamp.

4. The system of claim 2, wherein: the second sterilization lamp extends into an interior of the seed reactor vessel; and the second sterilization lamp is an ultraviolet-C light based sterilization lamp.

5. The system of claim 2, wherein the first sterilization lamp and the second sterilization lamp produce light centered at 254 nm.

6. The system of any one of claims 2 to 5, wherein: the production reactor vessel comprises: a vessel body having an opening, and a vessel lid sealingly closing the opening of the vessel body; the seed reactor vessel comprises: a seed vessel body having an opening, and a vessel lid sealingly closing the opening of the seed vessel body; the first sterilization lamp is connected to and supported by the vessel lid of the production reactor vessel; andthe second sterilization lamp is connected to and supported by the vessel lid of the seed reactor vessel.

7. The system of any one of claims 1 to 6, further comprising a water delivery assembly for providing input water to the seed reactor vessel and the production reactor vessel, the water delivery assembly comprising: a first water pump fluidly connected to at least one of the seed reactor vessel and the production reactor vessel, the first water pump being arranged to pump input water to the at least one of the vessels; and a second water pump fluidly connected to the seed reactor vessel and the production reactor vessel, the second water pump being arranged to pump wastewater out of the seed reactor vessel and the production reactor vessel.

8. The system of claim 7, wherein the water delivery assembly further comprises: at least one first flow-through water sterilizer in fluid communication with the first water pump for sterilizing the input water prior to introduction in the at least one of the vessels; and at least one second flow-through water sterilizer in fluid communication with the second water pump for sterilizing the wastewater expelled from the seed reactor vessel and the production reactor vessel.

9. The system of claim 8, further comprising at least one filter for filtering wastewater prior to sterilization.

10. The system of claim 9, wherein the at least one filter includes a submicron pore size filter.

11. The system of any one of claims 1 to 10, an aeration system operatively connected to the production reactor vessel, the aeration system comprising: a sparger disposed in the production reactor vessel; and an air pump fluidly connected to the sparger, during operation, the air pump causing air to flow into the production reactor vessel through the sparger to aerate contents in the production reactor vessel.

12. The system of claim 11, wherein the air pump is further fluidly connected to the seed reactor vessel for aerating contents in the seed reactor vessel during operation thereof.

13. The system of claim 11 or 12, further comprising a compressor system fluidly connected to the seed reactor vessel and the production reactor vessel for collecting humidity from air expelled from the vessels.

14. The system of any one of claims 1 to 13, further comprising an agitator disposed in the production reactor vessel for agitating contents of the production reactor vessel during operation.

15. The system of claim 14, wherein the agitator comprises: an impeller disposed in an interior of the production reactor vessel; and a motor disposed on an exterior of the production reactor vessel, the impeller being magnetically coupled to the motor for driving thereby.

16. The system of any one of claims 1 to 15, further comprising a plurality of sensors connected to the production reactor vessel, the plurality of sensors being arranged and configured to sense operating conditions in the production reactor vessel.

17. The system of claim 16, wherein the plurality of sensors include at least one of: a pH sensor; a dissolved oxygen (DO) sensor; a temperature sensor; and a camera.

18. The system of claim 16 or 17, wherein: the production reactor vessel comprises: a vessel body having an opening, and a vessel lid sealingly closing the opening of the vessel body; and at least one of the plurality of sensors is connected to and supported by the vessel lid.

19. The system of any one of claims 16 to 18, further comprising a computer-implemented system operatively connected to the plurality of sensors, the computer-implemented system including a human-machine interface (HMI) for providing information to an operator and receiving commands from the operator for controlling operation of the bioreactor system.

20. The system of any one of claims 1 to 19, further comprising at least one peristaltic pump fluidly connected between the seed reactor vessel and the production reactor vessel for pumping the seed inoculum to the production reactor vessel.

21. The system of any one of claims 1 to 20, further comprising a pH control system operatively connected to the production reactor vessel for managing pH levels of fermentation during operation.

22. A method for obtaining a microbial strain for use with the bioreactor system of claim 1 , the method comprising: i) culturing the microbial strain on a culture agar medium in presence of yeast extract, glucose, and peptone for a culture period of between about 18 hours and about 144 hours; and ii) fermenting the cultured microbial strain of step i) on a fermentation agar medium in presence of yeast extract, glucose, and peptone for a fermentation period of between about 18 hours and about 144 hours.

23. The method of claim 22, wherein step i) is performed at a pH of between about 5.5 and about 7.5.

24. The method of claim 22, wherein step ii) is performed at a pH of between about 5.5 and about 7.5.

25. The method of claim 22, wherein step i) is performed at a temperature of between about 20°C and about 30°C.

26. The method of claim 22, wherein step ii) is performed at a temperature of between about 20°C and about 30°C.

27. The method of claim 22, wherein the microbial strain is a fungal strain.

28. The method of claim 22, wherein the microbial strain is a mycelium.

29. A method for obtaining a microbial-based hydrolysate, the method comprising: i) drying the microbial strain obtained from the bioreactor system of claim 1; ii) heating the dried microbial strain of step i) to reduce RNA content; iii) hydrolyzing the microbial strain of step ii); iv) filtering and centrifugating the microbial strain of step iii); and vi) pasteurizing the microbial strain of step iv) to obtain a microbial -based hydrolysate.

30. The method of claim 29, wherein prior to step i), the microbial strain is rinsed with water at a temperature of between 10°C about and about 30°C.

31. The method of claim 29, wherein step ii) is carried out at a temperature of about 60°C for about 1 hour.

32. The method of claim 29, wherein step ii) is carried out using microwave.

33. The method of claim 29, wherein step iii) is carried out using acid, alkaline pH, heat, enzymes or fermentation.

34. The method of claim 29, wherein step iii) is carried out using proteases.

35. The method of claim 29, wherein the microbial strain is a fungal strain.

36. The method of claim 29, wherein the microbial strain is a mycelium.

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