Systems and methods for cultivation of methane-oxidizing microorganisms

Abstract

The present disclosure relates to bioreactor cascade systems and methods for the cultivation of microorganisms. In some embodiments, a bioreactor cascade system includes a plurality of bioreactors arranged in series, wherein each bioreactor is configured to receive a mixture of gaseous substrates necessary for the growth of microorganisms; a gas supply arrangement that delivers components of the nutrient medium in gaseous form into each bioreactor; and a gas phase recirculation mechanism that channels the exhaust gas mixture from a preceding bioreactor to a succeeding bioreactor.

Description

C1PRO.003WO PATENT SYSTEMS AND METHODS FOR CULTIVATION OF METHANE-OXIDIZING MICROORGANISMS INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 CFR 1.57. This application claims priority to U.S. Provisional Patent Application No. 63 / 561,697, filed March 5, 2024, which is hereby incorporated by reference in its entirety. BACKGROUND

[0002] The present disclosure relates to gas fermentation reactors, systems, and processes that utilize gaseous substrates for the production of various products, including single-cell protein, biofuels, and organic acids.

[0003] Gas-feed bioreactors have been widely studied for the production of single- cell protein (SCP) as well as biofuels, biopolymers, and pharmaceutical substances based on C1-compounds, including methane fermentation for SCP production, methane conversion to biofuels, methanol and formaldehyde production, methane conversion to biopolymers, or methane conversion to pharmaceutical substances.

[0004] Methane is a C1 compound that can be used as a substrate for SCP production. Various types of bioreactors have been developed for this purpose, including loop and jet fermenters. For example, a loop fermenter for methane fermentation is described in US Patent No. 10,184,103, describing U-shaped or nozzle U-loop fermenters and methods of fermentation. Jet fermenters are described in US Patent No. 6,558,898. The efficiency of methane fermentation for SCP production can be improved by optimizing the gas-liquid mixing system in the bioreactor, as described in US Patent Application 2017 / 0281322.

[0005] Methane can also be converted to higher-order hydrocarbons, such as liquid fuels, through processes such as gas-to-liquids (GTL) and Fischer-Tropsch (F-T) synthesis. Several patents have been filed for these processes, including US Patent No.9,457,405 for a GTL process using a plasma reactor, and US Patent No.10,308,789 for a F-T process using a cobalt-based catalyst.

[0006] Methanol and formaldehyde are C1 compounds that are commonly used as feedstocks for the production of various chemicals and materials. Methanol can be produced from methane through a process known as methanol synthesis, as described in US Patent No. 9,321,022. Formaldehyde can be produced from methanol through a process known as the silver-catalyzed oxidation of methanol, as described in US Patent No.10,119,556.

[0007] Methane can also be converted to biopolymers, such as polyhydroxyalkanoates (PHA), through microbial fermentation. A high productivity methane fermentation processes is described in US Patent No.10,934,566.

[0008] Methane can also be used as a substrate for the production of pharmaceutical substances, such as antibiotics and enzymes. For example, a methane-fed bioreactor for the production of aminoglycoside antibiotics is described in US Patent No. 7,449,323. SUMMARY

[0009] The systems, compositions, and methods disclosed herein each have several aspects, no single one of which is solely responsible for their desirable attributes. Without limiting the scope of the claims, some embodiments are described below. Numerous additional embodiments are also contemplated, including embodiments that have fewer, additional, and / or different components, steps, features, objects, benefits, and advantages. The components, aspects, and steps may also be arranged and ordered differently.

[0010] Some embodiments provided herein relate to bioreactor cascade systems for cultivation of microorganisms. In some embodiments, the systems include a plurality of bioreactors arranged in series. In some embodiments, the series is referred to as a cascade. In some embodiments, each bioreactor is configured to receive a mixture of gaseous substrates necessary for the growth of microorganisms. In some embodiments, the systems are characterized by a gas supply arrangement that delivers components of the nutrient medium in gaseous form into each bioreactor, and a gas phase recirculation mechanism designed to channel the exhaust gas mixture from a preceding bioreactor to a succeeding bioreactor, while the first bioreactor in the series is distinctly provided with an initial supply of natural gas or an alternative gaseous carbon source, along with pressurized air, oxygen, or oxygen-enriched air. In some embodiments, the gaseous carbon source is biogas, methane, or an associated gas. Insome embodiments, each bioreactor in the cascade is operatively connected to ensure the transfer of gases therethrough. In some embodiments, the systems include an outlet for the exhaust gases at a predetermined stage in the cascade, referred to as the K-step. In some embodiments, the number of stages is determined based on the operational mode. In some embodiments, the number of stages is not less than two.

[0011] In some embodiments, each bioreactor in the series is configured to operate at a higher pressure than the preceding bioreactor, thereby incrementally increasing the pressure throughout the cascade. In some embodiments, the incremental increase in pressure enhances the solubility and mass transfer rates of gases with low solubility.

[0012] In some embodiments, the systems are configured to enrich the gas phase with oxygen at one or more selected stages within the cascade, the quantity of oxygen introduced at each said stage being dynamically determined based on the gas composition outcomes from the preceding stage(s). In some embodiments, enrichment is independent of any additional pressure application in subsequent stages.

[0013] In some embodiments, a specific energy input at each stage is managed to maintain the required level of mass transfer efficiency. In some embodiments, an energy management negates the necessity for supplemental oxygen or additional pressure in subsequent stages of the cascade system.

[0014] In some embodiments, the systems include a gas preparation module configured to adjust the carbon dioxide content or enrich the gas mixture with pure oxygen, thereby supporting an efficient ratio of gases in the gas mixture.

[0015] In some embodiments, the systems include a gas supply system. In some embodiments, the gas supply system includes a controller for adjusting the composition of the gas mixture to optimize the partial pressures of oxygen or carbon dioxide at each stage of the cascade.

[0016] In some embodiments, the systems include a jet-stream bioreactor configuration for each stage within the cascade. In some embodiments, one or more of the plurality of bioreactors is equipped with a jet-stream parameters control system. In some embodiments, the jet-stream parameters control system uses advanced automated monitoring and artificial intelligence, including machine learning algorithms, to dynamically adjust the velocity, pressure, and composition of the liquid-gas jets introduced into the liquid medium.In some embodiments, the jet-stream parameters control system is configured to optimize the dissolution rates of methane and oxygen by continuously adapting jet parameters based on real-time analysis of microbial growth conditions, gas solubility, and mass transfer efficiency. In some embodiments, the jet-stream parameters control system is configured to maintain operational safety by maintaining gas mixture compositions within safe explosive limits, as defined by the Gibbs-Roseboom diagram, and optimizes energy consumption by adjusting jet parameters to achieve maximal substrate utilization and minimal energy expenditure.

[0017] In some embodiments, the systems are configured as a modular design. In some embodiments, the modular design includes a configuration where each bioreactor and associated gas supply and recirculation components are configured as standalone modules that can be independently operated or seamlessly integrated into the systems. In some embodiments, the modular design allows for expansion or reduction of the system’s capacity to accommodate different production scales and operational requirements, facilitating the customization of the bioreactor cascade to specific process needs or to experiment with different microbial cultures and gaseous substrates.

[0018] In some embodiments, the systems include a waste gas utilization module designed to capture and process exhaust gases from the cascade for secondary applications. In some embodiments, the waste gas utilization module separates and purifies methane, carbon dioxide, and other gaseous byproducts from the exhaust stream, rendering them suitable for use in energy generation systems, such as combined heat and power (CHP) units, or as substrates in additional fermentation processes, including but not limited to manure methanation or biohydrogen production. In some embodiments, the waste gas utilization module includes technologies for gas cleaning, separation, and compression, tailored to meet the specifications required by downstream processes or energy systems, thereby enhancing the overall sustainability and resource efficiency of the bioreactor systems, and contributing to a circular economy model by transforming waste gases into valuable resources.

[0019] In some embodiments, the systems include a monitoring system for real- time analysis of gas composition, specific electricity input, and mass-transfer coefficients, to maintain safe operational parameters and achieve efficient microbial growth with a reduced carbon footprint.

[0020] In some embodiments, the optimal ratio of different gases is determined based on the specific requirements for cultivation of different microorganisms, including those used for the production of single-cell proteins, biofuels, biopolymers, or pharmaceutical substances.

[0021] In some embodiments, the systems are configured to be adjusted to optimize an aim function, such as carbon footprint, gaseous substrate utilization coefficient, specific energy input, or overall system productivity.

[0022] In some embodiments, the systems include a gas recycling module designed to ensure the explosion safety of the technological process by controlling the composition of the gas phase and maintaining safe concentrations of methane and oxygen.

[0023] In some embodiments, the systems are configured to achieve high process productivity, such as a production rate of at least about 4 kg of absolutely dry matter per hour per cubic meter of the bioreactor’s working volume.

[0024] Some embodiments disclosed herein relate to bioreactor cascade systems for the cultivation of microorganisms. In some embodiments, the systems include a plurality of bioreactors arranged in series, a gas supply arrangement that delivers carbon and energy sources in gaseous form into each bioreactor, and a gas phase recirculation mechanism that channels an exhaust gas mixture from a preceding bioreactor to a succeeding bioreactor. In some embodiments, each bioreactor is configured to receive a mixture of gaseous substrates necessary for growth of microorganisms.

[0025] In some embodiments, each bioreactor in the series is operatively connected to ensure the transfer of gases therethrough. In some embodiments, a first bioreactor in the series is configured to receive an initial supply of natural gas or an alternative gaseous carbon source, along with pressurized air, oxygen, or oxygen-enriched air. In some embodiments, the alternative gaseous carbon source is biogas, methane, or associated gas.

[0026] In some embodiments, the plurality of bioreactors arranged in series are configured to operate in a cascade. In some embodiments, the systems include an outlet for the exhaust gas mixture at a predetermined stage in the cascade. In some embodiments, the predetermined stage is determined based on an operational mode. In some embodiments, the cascade includes at least two stages.

[0027] In some embodiments, each bioreactor in the series is configured to operate at a higher pressure than the preceding bioreactor. In some embodiments, the systems are configured to enrich the gas phase with oxygen at one or more selected stages within the series. In some embodiments, a quantity of oxygen introduced at each said stage is dynamically determined based on gas composition outcomes from at least one preceding stage. In some embodiments, the enrichment is independent of any additional pressure application in subsequent stages.

[0028] In some embodiments, the systems are configured to maintain the required level of mass transfer efficiency. In some embodiments, the system is configured such that the energy management negates the necessity for supplemental oxygen or additional pressure in subsequent stages of the cascade system.

[0029] In some embodiments, the systems include a gas preparation module configured to adjust the carbon dioxide content or enrich the gas mixture with pure oxygen. In some embodiments, the gas preparation module maintains an efficient ratio of gases in the gas mixture. In some embodiments, the gas supply arrangement includes a controller for adjusting the composition of the gas mixture to optimize the partial pressures of oxygen or carbon dioxide at each stage of the cascade.

[0030] In some embodiments, the systems include a jet-stream bioreactor configuration for each stage within the cascade. In some embodiments, the jet-stream bioreactor is equipped with a jet-stream parameters control system. In some embodiments, the jet-stream parameters control system uses advanced automated monitoring and artificial intelligence to dynamically adjust the velocity, pressure, and composition of the liquid-gas jets introduced into the liquid medium. In some embodiments, the jet-stream parameters control system is configured to optimize dissolution rates of methane and oxygen by continuously adapting jet parameters based on real-time analysis of microbial growth conditions, gas solubility, and mass transfer efficiency. In some embodiments, the jet-stream parameters control system maintains gas mixture compositions within safe explosive limits, as defined by the Gibbs-Roseboom diagram. In some embodiments, the jet-stream parameters control system is configured to optimize energy consumption by adjusting jet parameters to achieve maximal substrate utilization and minimal energy expenditure. In some embodiments, each bioreactorand gas supply and recirculation component are configured as standalone modules that can be independently operated or integrated into the series.

[0031] In some embodiments, the systems include a waste gas utilization module configured to capture and process exhaust gases from the cascade for secondary applications. In some embodiments, the waste gas module separates and purifies methane, carbon dioxide, and other gaseous byproducts from the exhaust stream. In some embodiments, the systems include a monitoring system for real-time analysis of gas composition, specific electricity input, and mass-transfer coefficients.

[0032] In some embodiments, systems are configured to be adjusted to optimize an aim function, such as carbon footprint, gaseous substrate utilization coefficient, specific energy input, or overall system productivity. In some embodiments, the systems include a gas recycling module configured to control a composition of a gas phase and to maintain safe concentrations of methane and oxygen. In some embodiments, the systems are configured to produce at least 4 kg of absolutely dry matter per hour per cubic meter of working volume.

[0033] Some embodiments provided herein relate to methods for the cultivation of microorganisms. In some embodiments, the methods includes providing a bioreactor cascade system that includes a plurality of bioreactors arranged in series, providing a first bioreactor in the series with an initial supply of natural gas or an alternative gaseous carbon source, delivering a nutrient medium in gaseous form into each bioreactor via a gas supply arrangement, channeling the exhaust gas mixture from a preceding bioreactor to a succeeding bioreactor via a gas phase recirculation mechanism, and letting out exhaust gases at a predetermined stage in the cascade. In some embodiments, the predetermined stage is determined based on the operational mode but is at least a second stage. In some embodiments, each bioreactor is configured to receive a mixture of gaseous substrates necessary for the growth of microorganisms, such as biogas, methane, or an associated gas, along with pressurized air, oxygen, or oxygen-enriched air. In some embodiments, each bioreactor in the cascade is operatively connected to ensure the transfer of gases therethrough.

[0034] In some embodiments, the methods include incrementally increasing the pressure throughout the cascade to enhance solubility and mass transfer rates of gases with low solubility. In some embodiments, the methods include enriching the gas phase with oxygen at one or more selected stages within the cascade. In some embodiments, the methods includedynamically determining a quantity of oxygen introduced at each said stage based on the gas composition outcomes from the preceding stage(s). In some embodiments, the oxygen enrichment is independent of any additional pressure application in subsequent stages.

[0035] In some embodiments, the methods include managing the specific energy input at each stage to maintain the required level of mass transfer efficiency. In some embodiments, the energy management negates the necessity for supplemental oxygen or additional pressure in subsequent stages of the cascade system. In some embodiments, the methods include adjusting carbon dioxide content or enriching the gas mixture with pure oxygen, thereby supporting an efficient ratio of gases in the gas mixture. In some embodiments, the methods include adjusting the composition of the gas mixture to optimize the partial pressures of oxygen or carbon dioxide at each stage of the cascade.

[0036] In some embodiments, the methods include integrating a jet-stream bioreactor configuration for each stage within the cascade. In some embodiments, the bioreactors are equipped with a jet-stream parameters control system. In some embodiments, the methods include monitoring the bioreactors and dynamically adjusting the velocity, pressure, and composition of the liquid-gas jets introduced into the liquid medium. In some embodiments, the methods include optimizing dissolution rates of methane and oxygen by continuously adapting jet parameters based on real-time analysis of microbial growth conditions, gas solubility, and mass transfer efficiency. In some embodiments, the methods include maintaining gas mixture compositions within safe explosive limits. In some embodiments, the methods include optimizing energy consumption by adjusting jet parameters to achieve maximal substrate utilization and minimal energy expenditure.

[0037] In some embodiments, the methods include customizing the bioreactor cascade to specific process needs or to experiment with different microbial cultures and gaseous substrates.

[0038] In some embodiments, the methods include capturing and processing exhaust gases from the cascade for at least one secondary application. In some embodiments, the methods include separating and purifying methane, carbon dioxide, or other gaseous byproducts from the exhaust stream, and rendering the methane, carbon dioxide, or other gaseous byproducts suitable for use in energy generation systems, or as substrates in additional fermentation processes.

[0039] In some embodiments, the methods include monitoring the series of bioreactors for real-time analysis of gas composition, specific electricity input, or mass-transfer coefficients. In some embodiments, the methods include adjusting the configuration and operation of the cascade system to optimize an aim function, such as carbon footprint, gaseous substrate utilization coefficient, specific energy input, or overall system productivity.

[0040] In some embodiments, the methods include adjusting the composition of the gas phase and maintaining safe concentrations of methane and oxygen. In some embodiments, the methods include producing at least 4 kg of absolutely dry matter per hour per cubic meter of working volume. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear. In addition to the features described herein, additional features and variations will be readily apparent from the following descriptions of the drawings and exemplary embodiments. It is to be understood that these drawings depict typical embodiments, and are not intended to be limiting in scope.

[0042] Figure 1 is a Gibbs-Roseboom diagram for three components: oxygen, methane, and nitrogen.

[0043] Figure 2 schematically illustrates an enhanced bioreactor cascade system with integrated gas management for microbial cultivation.

[0044] Figure 3 is a graph that illustrates dynamic changes in essential parameters throughout the fermentation process. DETAILED DESCRIPTION

[0045] The foregoing and other aspects of the present disclosure will now be described in more detail with respect to the description and methodologies provided herein. This description is not intended to be a detailed catalogue of all the ways in which the embodiments of the present disclosure may be implemented, or of all the features that may beadded to the present disclosure. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein, which do not depart from the instant disclosure, will be apparent to those skilled in the art in light of the instant detailed description, figures, and claims. Hence, the following specification is intended to illustrate some particular embodiments, and not to exhaustively specify all permutations, combinations, and variations thereof.

[0046] All patents, patent applications, and other publications, including all sequences disclosed within these references, referred to herein are expressly incorporated herein by reference, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. All documents cited are, in relevant part, incorporated herein by reference in their entireties for the purposes indicated by the context of their citation herein. However, the citation of any document is not to be construed as an admission that it is prior art with respect to the present disclosure.

[0047] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0048] The present disclosure relates to novel fermentation cascade systems that (1) improve gas / liquid mixing and facilitate efficient mass transfer, while (2) minimizing the carbon footprint and (3) ensuring the safety of the process. The present disclosure also relates to the (4) efficient utilization of gaseous substrates and the (5) optimization of energy consumption in gas fermentation systems. The present disclosure has applications in themicrobiological industry and can be used for large-scale production of various valuable products.

[0049] The present disclosure relates to systems and methods of gas fermentation to address the challenges associated with large-scale production of single-cell protein (SCP), biopolymers (including, for example, PHA - polyhydroxyalkanoates), enzymes (including, for example, lipase) and other high-added value products from C1 substrates (including, for example, natural gas, biogas, or other carbon sources). The current gas-fermentation methods and apparatus mainly rely on single loop or single jet-stream fermenters, but these methods produce a significant amount of carbon dioxide, which contributes to the carbon footprint of the process. Additionally, loop fermenters require the use of pure oxygen for efficient operation and may form explosive gas mixtures during the process, which further complicates large-scale SCP production. One of the key challenges in gas-fermentation systems is energy efficiency. Methane and oxygen are low solubility gases and classical fermentation systems consume a lot of energy to dissolve these gases. The disclosed systems and methods of gas fermentation are designed to address this issue by optimizing the gas-liquid mixing system, which reduces the energy required to dissolve the gases and improves the efficiency of the fermentation process.

[0050] Classical methane fermentation systems used in SCP production produce carbon dioxide as a byproduct. The equation for the production of carbon dioxide during methane fermentation is: CH4 + 2O2 → CO2 + 2H2O (1) For every ton of SCP produced using the classical fermentation scheme, approximately 1.7 tons of CO2are output. This contributes significantly to the carbon footprint of the process, and reducing this output is one of the main priorities in the development of technologies for the microbiological industry.

[0051] Another important parameter in gas-fermentation is the coefficient of substrate utilization. Classical fermentation schemes and methods cannot provide full methane and oxygen utilization, and the coefficient of utilization of both gases cannot be more than 70%. To address this issue, the disclosed system and method of gas fermentation optimize the gas-liquid mixing system, which improves the efficiency of gas utilization and increases the coefficient of substrate utilization.

[0052] The novel fermentation cascade methods and systems of the present disclosure are designed to significantly reduce the carbon footprint during the fermentation process. This is achieved by optimizing the gas / liquid mixing system in the cascade process, which maximizes the mass transfer rate between the gas and liquid phases. This improves the efficiency of the fermentation process and reduces the amount of unreacted gases that contribute to the carbon dioxide output. Additionally, the cascade systems are designed to recirculate unreacted gases back into the reactor, further reducing the carbon footprint.

[0053] Furthermore, the novel cascade systems address the safety concerns associated with the use of natural gas, oxygen, and nitrogen in the fermentation process. The optimized gas / liquid mixing systems minimize the risk of explosive gas mixtures forming in the reactor, ensuring the safety of the process.

[0054] The present disclosure also provides efficient methods for utilizing gaseous substrates, such as methane, in the fermentation process. The cascade systems allow for multiple stages of fermentation, ensuring that the gaseous substrate is efficiently utilized by the microorganisms, leading to improved product yields and reduced waste. Additionally, the cascade systems allow for the efficient use of energy, as the system is designed to recycle waste heat and optimize energy consumption.

[0055] Overall, the present disclosure provides a significant improvement in the field of gas fermentation systems, providing a more efficient, safe, and sustainable method for producing single-cell protein and other valuable products. The use of the novel cascade systems allows for improved product yields and reduced carbon footprint, making it a valuable tool for the microbiological industry. Definitions

[0056] Although the following terms are believed to be well understood by one of skill in the art, the following definitions are set forth to facilitate understanding of the presently disclosed subject matter.

[0057] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art.

[0058] As used herein, the terms “a” or “an” or “the” may refer to one or more than one. For example, “a” marker can mean one marker or a plurality of markers.

[0059] As used herein, the term “about,” when used in reference to a measurable value such as an amount of mass, dose, time, temperature, and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0060] As used herein, the term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0061] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0062] As used herein, the term “consists essentially of” (and grammatical variants thereof), as applied to the compositions and methods of the present disclosure, means that the compositions / methods may contain additional components so long as the additional components do not materially alter the composition / method.

[0063] As used herein, the term “bioreactor” refers to a controlled environment system designed for the cultivation of biological organisms, typically microorganisms or cells, under specific conditions conducive to their growth, proliferation, and desired metabolic activities.

[0064] As used herein, the term “bioreactor cascade system” refers to a plurality of bioreactors arranged in series. In some embodiments, a process for the cultivation of microorganisms may progress in a single direction along the series in the bioreactor cascade system.

[0065] As used herein, the term “stage” refers to a step of a process for the cultivation of microorganisms, such as may be implemented on a bioreactor cascade system. A stage may include a series of chemical reactions which take place in a bioreactor. In some embodiments, each “stage” includes a separate bioreactor of the plurality of bioreactors arranged in series. In some embodiments, two or more stages take place using the same bioreactor.

[0066] As used herein, the term “absolutely dry matter” also referred to as “dry matter,” refers to a measure of the mass of a completely dried substance, such as the total solid content of a material produced in a bioreactor or bioreactor system after all moisture (e.g., water) has been completely removed. For example, 100% of all moisture can be removed, or more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of moisture can be removed.

[0067] (1) Gas-liquid mass transfer is the main factor limiting the efficiency of gas fermentation due to the poor solubility of gaseous substrates in liquids. The rate of mass transfer can be described by the Volumetric mass transfer rate equation: ^^^^ െ ^^ ^^^^ൌ ^^^^^^^^ ∗ െ^^^^ (2)where: ௗെ ே^ೃௗ௧is the rate at which the gaseous substrate is transferred to the liquid phase; ^^^^^ is the volumetric mass transfer coefficient which consists of the liquid side mass transfer coefficient ^^^and the specific mass transfer surface area, ^^ ^^ ∗ is the saturation concentration of the gas in the liquid (i.e., the solubility) which isproportional to the partial pressure of the gaseous substrate (O2 or CH4) ^^^is the actual gas concentration in the liquid, which is the difference between ^^ ∗ and the actual gas concentration in the liquid (i.e., ^^^ ∗ െ^^^^ is the mass transferdriving force).

[0068] Under pure mass-transfer limited conditions ^^^ ^ O.^^ோis the wetted volume of the reactor which is the sum of gas volume and liquid volume.

[0069] To improve the efficiency of mass transfer, it is necessary to either increase ^^^^^ or the driving force. Increasing the driving force by using higher pressure is efficient but expensive as it requires the compression of gas. Therefore, it is more preferable to increase ^^^and / or ^^.

[0070] The specific mass transfer surface area, ^^, has a simple relationship with the gas holdup, ε, and the average bubble radius, ^^^. This relationship can be described as:3ε ^^ ൌ ீ(3) ^^^where: where ^^ - specific mass transfer surface area, εீ– gas holdup, ^^^- bubble size

[0071] From equation (2), it is clear that the specific mass transfer area can be increased by an increase in gas holdup, εீ, or a decrease in bubble size, ^^^, or a combination of both.

[0072] In summary, to improve the efficiency of gas fermentation, it is necessary to improve the rate of mass transfer by increasing ^^^and / or ^^. Gas holdup and bubble size are factors that can be manipulated to increase the specific mass transfer surface area, ^^.

[0073] High-efficient jet-stream bioreactors produce high-turbulent two-phase medium to increase specific mass transfer surface area, ^^ .

[0074] (2) Minimizing the carbon footprint in the context of bioreactor systems, particularly those that utilize methane and carbon dioxide as part of the fermentation process, involves reducing the release of these gases into the atmosphere and optimizing energy usage throughout the process.

[0075] Conventional bioreactors often operate at a suboptimal efficiency in terms of gas utilization, leading to the release of exhaust gases that still contain significant amounts of methane and carbon dioxide. These potent greenhouse gases contribute to the carbon footprint of the process when vented into the atmosphere.

[0076] Filtration or scrubbing of carbon dioxide from exhaust gases is less feasible when the concentration of CO2is low, as the energy and technological requirements for extraction become disproportionately high relative to the yield. Therefore, processes that emit exhaust with low CO2 concentrations are challenging to make carbon-neutral due to the additional energy (and associated emissions) required for CO2capture.

[0077] Similar to carbon dioxide, reusing methane from exhaust gases is inefficient when its concentration is low. The process of capturing and purifying methane to a usable level is energy-intensive and not cost-effective for low-concentration streams. Consequently, it is typically released, contributing to the overall carbon footprint.

[0078] High energy consumption inherently increases the carbon footprint of bioreactor operations. This is because the energy used for gas compression, mixing, and other process requirements is often generated from carbon-emitting sources.

[0079] To address these issues, the cascade bioreactor systems and methods may, in some embodiments, implement one or more strategies described below:

[0080] Maximized Gas Utilization: By efficiently using the gaseous substrates in each stage of the cascade, the system minimizes the methane and carbon dioxide content in the exhaust gases. This is achieved through better gas-liquid mass transfer and the stepwise utilization of gases, reducing the volume that must be treated or released.

[0081] Recirculation of Exhaust Gases: The cascade system recycles exhaust gases, using them as an input for the subsequent stage. This recycling reduces the need to vent methane and carbon dioxide, cutting down the emission of greenhouse gases.

[0082] Reduced Need for Gas Purification: By maintaining higher concentrations of CO2 between the steps through controlled consumption, the cascade system allows for more efficient capture, making the process more economically and technologically viable.

[0083] Energy Efficiency: The cascade system is designed to operate more energy- efficiently by reducing the energy required for gas compression and optimizing operational parameters. This decreases the overall energy demand and the associated carbon emissions from energy sources.

[0084] By integrating these strategies, the cascade bioreactor systems and methods disclosed herein may not only improve the efficiency of microbial growth and product yield but also significantly reduce the carbon footprint compared to conventional bioreactor systems, for example, up to 5 times more. This approach aligns with the broader objectives of sustainable industrial practices and the reduction of greenhouse gas emissions.

[0085] (3) Ensuring the safety of the process. Methane-based fermentation is a complex process that requires careful consideration of safety conditions to prevent explosive mixtures from forming. In the case of U-loop bioreactors, the technology developers have chosen a safe operation area above the MA line, with a maximum concentration of methane of 6%. The safe operation area is determined by the explosive limits of the fuel-oxidizer-inert component system, which can be calculated using the Gibbs-Roseboom triangular diagram.

[0086] Figure 1 illustrates a Gibbs-Roseboom triangular diagram. The diagram includes three components: Oxygen, Methane, and Nitrogen. Oxygen is the oxidizer required for the combustion of methane, which is the fuel. Nitrogen is an inert gas that does not support combustion or react with the other components under normal conditions. The chart indicates regions where mixtures are flammable (LEL - Lower Explosive Limit and UEL - Upper Explosive Limit) and the stoichiometric line, which represents the ideal mixing ratio for complete combustion of methane with oxygen. The LOC (Limit of Combustion) represents the concentration below which combustion cannot occur.

[0087] In the cascade fermentation system, managing the concentrations of oxygen and methane is crucial for both the process efficiency and safety. Ensuring that the gas mixtures at each stage of the system remain outside of the flammable and explosive regions is essential for safe operation. Nitrogen may be used within the fermentation system to control the oxygen concentration and prevent the formation of flammable mixtures, which can be a hazard especially in enclosed spaces where gases are handled. The fermentation system may have monitoring and control mechanisms to ensure that the gas mixtures remain within safe and efficient operating ranges, taking into account the information presented in the flammability diagram.

[0088] The explosive limits of the methane-oxidizing mixture are in the range of 5.0-61.0% by volume. With an excess of oxygen, the cultivation process is limited by the rate of dissolution of methane, and the spent gas-air mixture with a methane content of up to 3% is released into the atmosphere, contributing to environmental pollution.

[0089] In contrast, jet-stream bioreactors operate in a safe operation area below the MB line, with a safe operation area limited to 8% oxygen in the entire range below the GW line. This is due to the difficulties in regulating the ratios of the three components of the gas mixture. The explosive limits of the fuel-oxidizer-inert component system can also be calculated using the Gibbs-Roseboom triangular diagram. With an excess of methane, the cultivation process is limited by the rate of oxygen dissolution, and the spent gas-air mixture with a natural gas content of up to 30%, oxygen of 8% (the rest is nitrogen and carbon dioxide) enters the drying stage as fuel or power generation.

[0090] To determine the safe operating range of the fuel-oxidizer-inert component system, the following equations may be used:

[0091] For the U-loop bioreactor: Explosive limits of the methane-oxidizing mixture are in the range of 5.0-61.0% by volume. A safe operation area is above the MA line, with a maximum concentration of methane of 6%.

[0092] For the jet-stream bioreactor: Safe operation area is below the MB line, with a safe operation area limited to 8% oxygen in the entire range below the GW line. Explosive limits of the methane-oxidizing mixture can be calculated using the Gibbs-Roseboom triangular diagram. Overall, the safe operation of methane-based fermentation in U-loop and Jetstream bioreactors requires careful consideration of the explosive limits of the fuel-oxidizer- inert component system. The selection of a safe operation area can be determined by using the Gibbs-Roseboom triangular diagram, and the growth rate of methane-oxidizing microorganisms is limited by the rate of dissolution of methane or oxygen.

[0093] The disadvantage of the operation of U-loop and jet-stream bioreactors is that in both cases, one of the components of the gas mixture is in the limit, which can lead to inefficient use of energy. This is because the consumption of one component limits the other component, and an unreasonably large amount of energy is spent on the other component for its dissolution.

[0094] For example, in case of U-loop bioreactor if the concentration of methane is increased above the safe limit, there is a risk of an explosive mixture forming. Conversely, if the concentration of oxygen is increased, it will not lead to an explosive mixture, but it will not increase the growth rate of methane-oxidizing microorganisms. Instead, the excess oxygen will simply dissolve into the liquid medium, wasting energy.

[0095] Overall, the safe operation of U-loop and Jet-stream bioreactors requires careful consideration of the ratios of the three components of the gas mixture. An unbalanced ratio can lead to inefficient use of energy and environmental pollution. Therefore, it is essential to optimize the gas mixture ratio to maximize the growth rate of methane-oxidizing microorganisms while ensuring safety conditions.

[0096] (4) Efficient utilization of gaseous substrates. The cascade bioreactor system presents a significant advancement over conventional bioreactor systems, particularly in its efficient utilization of gaseous substrates like methane and oxygen. An in-depth explanation of how the cascade system improves upon traditional methods follows:

[0097] In typical bioreactor systems, the utilization rates of gaseous substrates are limited to 50-60% efficiency. This limitation is largely due to safety constraints; to maintain a non-explosive mixture of methane and oxygen, the proportions of these gases must align with the safe zones defined by the Gibbs-Roseboom diagram for methane-oxygen-inert gas mixtures. As a result, either methane or oxygen is supplied in excess to avoid entering the explosive range, leading to significant inefficiencies, as the excess gas is ultimately vented to the atmosphere in the exhaust.

[0098] In some embodiments, the cascade system transcends these limitations through several methods, each aiming to maximize substrate utilization up to 99% efficiency. Exemplary methods are further described below.

[0099] Pressure Increment Method: By increasing the pressure in each subsequent step of the cascade, the solubility of the gases in the liquid medium is enhanced, thus improving the mass transfer rate. Pros: Higher pressures generally lead to better gas solubility, thus allowing for greater substrate utilization. Cons: Increased pressure requires stronger, more expensive equipment and can lead to higher operational costs. The system’s complexity increases with the need for precise pressure control.

[0100] Oxygen Enrichment Method: This method involves adding pure oxygen to increase the partial pressure of oxygen in the mixture on each next steps of cascade, which can drive the reaction towards higher substrate consumption. Pros: Targeted addition of oxygen can precisely control the oxidation process and maximize the yield of the desired product. It can significantly reduce the volume of methane required, thus lowering the risk of reaching explosive gas concentrations. Cons: The cost of pure oxygen can be high, impacting the overall economic feasibility. Managing high oxygen levels requires careful control to avoid toxicity to the microorganisms and corrosion of the equipment.

[0101] Energy Input Method: By increasing the energy input, the system can enhance the mass transfer coefficient, which is the efficiency of gas transfer from the gaseous to the liquid phase and compensate methane or / and oxygen shortages to maintain productivity at a given level. Pros: Improved mass transfer rates can lead to increased microbial activity and product formation. This method allows for finer control over the fermentation process without altering the gas composition. Cons: Higher energy input translates to increased costsand possibly additional carbon footprint of the system. It may require sophisticated control systems to manage the energy distribution effectively.

[0102] Each method addresses the core challenge of maximizing gas substrate utilization while maintaining safe operation within the explosive limits. In some embodiments, by combining these approaches, the cascade system offers a versatile and efficient solution to gas fermentation, significantly improving upon the inefficiencies of conventional systems. However, the choice between these methods—or a combination thereof—will depend on specific process requirements, economic considerations, and safety regulations.

[0103] (5) Optimization of energy consumption. Methane and oxygen are low solubility gases and classical fermentation systems consume a lot of energy to dissolve these gases. In some embodiments, proposed systems and methods of gas fermentation are designed to address this issue by optimizing the gas-liquid mixing system, which reduces the energy required to dissolve the gases and improves the efficiency of the fermentation process. The optimization of energy consumption in a cascade gas fermentation system fundamentally rethinks how gases are introduced and utilized within a series of bioreactors. The goal is to decrease the energy demand required for dissolving low solubility gases like methane and oxygen, which is a significant energy sink in classical fermentation systems.

[0104] In standard fermentation setups, each bioreactor requires the gaseous substrates to be compressed individually to achieve sufficient partial pressures for dissolving the gases into the liquid medium. This process is energetically expensive due to the following factors. First, compressing gases to the high pressures necessary for solubilization is energy intensive. Second, each bioreactor operates independently, meaning that the compression energy invested in the gas for one reactor cannot be reused in another. Third, the energy expended in compressing the gas does not contribute to the fermentation once the gas is released and vented as exhaust.

[0105] The cascade gas fermentation systems and methods may introduce several innovations to optimize energy consumption. For example, instead of compressing gases for each bioreactor, the cascade system compresses the gas mixture once. This compressed gas is then sequentially used in the following bioreactor stages. This leverages the thermodynamic principle where the energy used to compress the gas can be partially retained in the gas itself as increased potential energy. Additionally, the initial energy input for compression isamortized over multiple stages, reducing the total energy consumption per unit of biomass produced. Finally, the systems and methods may include gas recirculation, where exhaust gases from one bioreactor are not vented but are instead recirculated in one bioreactor circuit and into the next stage. Gas recirculation takes advantage of the remaining partial pressure and potential energy in the exhaust gases, thus requiring less additional energy for subsequent dissolution of gases. Bioreactor Cascade Systems

[0106] In one aspect, disclosed herein are bioreactor cascade systems for the cultivation of microorganisms. In some embodiments, the system includes a plurality of bioreactors arranged in series, where each bioreactor is configured to receive a mixture of gaseous substrates necessary for the growth of microorganisms.

[0107] In some embodiments, the system includes a gas supply arrangement that delivers components (including, for example, carbon or energy sources) of the nutrient medium in gaseous form into each bioreactor, and a gas phase recirculation mechanism that channels the exhaust gas mixture from a preceding bioreactor to a succeeding bioreactor. In some embodiments, the gas supply arrangement delivers carbon and energy sources in gaseous form into each bioreactor. In some embodiments, the system includes means for delivery of other components of the nutrient medium via a liquid medium.

[0108] In some embodiments, the system is characterized by a gas supply arrangement that delivers components of the nutrient medium in gaseous form into each bioreactor, and a gas phase recirculation mechanism designed to channel the exhaust gas mixture from a preceding bioreactor to a succeeding bioreactor, while the first bioreactor in the series is distinctly provided with an initial supply of natural gas or an alternative gaseous carbon source. In some embodiments, each bioreactor in the cascade is operatively connected to ensure the transfer of gases therethrough. Examples of alternative gaseous carbon sources include (but are not limited to) biogas, methane, and associated gas.

[0109] In some embodiments, the plurality of bioreactors arranged in series are configured to operate in a cascade. In some embodiments, the system includes an outlet for the exhaust gases at a predetermined stage in the cascade. In some embodiments, the cascade includes a number of stages, and the predetermined stage is based on the operational mode. Insome embodiments, the cascade includes at least two stages. In some embodiments, the cascade includes two, three, four, five, six, seven, eight, nine, ten, or more stages, or a range constructed from any of the aforementioned values. In some embodiments, the system includes an outlet for the exhaust gases at a predetermined stage in the cascade, referred to as the K- step, where the number of stages is determined based on the operational mode but is not less than two.

[0110] In some embodiments, each bioreactor in the series is configured to operate at a higher pressure than the preceding bioreactor. In some embodiments, the pressure is thereby incrementally increased throughout the cascade to enhance the solubility and mass transfer rates of gases with low solubility.

[0111] In some embodiments, the system is further adapted to enrich the gas phase with oxygen at one or more selected stages within the cascade. In some embodiments, the quantity of oxygen introduced at each said stage is dynamically determined based on the gas composition outcomes from the preceding stage(s). In some embodiments, such enrichment is independent of any additional pressure application in subsequent stages.

[0112] In some embodiments, the system is configured such that a specific energy input at each stage is managed to maintain the required level of mass transfer efficiency. In some embodiments, the specific energy input at each stage is managed to maintain the required level of mass transfer efficiency, and this energy management negates the necessity for supplemental oxygen or additional pressure in subsequent stages of the cascade system. Thus, in some embodiments, the system is configured such that the energy management negates the necessity for supplemental oxygen or additional pressure in subsequent stages of the cascade system

[0113] In some embodiments, the system further includes a gas preparation module configured to adjust the carbon dioxide content and / or enrich the gas mixture with pure oxygen, thereby supporting an efficient ratio of gases in the gas mixture.

[0114] In some embodiments, the gas supply system or arrangement includes a controller for adjusting the composition of the gas mixture to optimize the partial pressures of oxygen and / or carbon dioxide at each stage of the cascade.

[0115] In some embodiments, the system further includes a jet-stream bioreactor configuration for each stage within the cascade. In some embodiments, the system is furtherenhanced by integrating a jet-stream bioreactor configuration for each stage within the cascade. In some embodiments, the bioreactors are equipped with a jet’s parameters control system. In some embodiments, this control system (the jet control system) utilizes advanced automated monitoring and artificial intelligence, including machine learning algorithms, to dynamically adjust the velocity, pressure, and composition of the liquid-gas jets introduced into the liquid medium. In some embodiments, the control system is specifically designed to optimize the dissolution rates of methane and oxygen by continuously adapting jet parameters based on real-time analysis of microbial growth conditions, gas solubility, and mass transfer efficiency. In some embodiments, the control system further ensures operational safety by maintaining gas mixture compositions within safe explosive limits, as defined by the Gibbs-Roseboom diagram, and / or optimizes energy consumption by adjusting jet parameters to achieve maximal substrate utilization and minimal energy expenditure.

[0116] In some embodiments, the system is characterized by its modular design. In some embodiments, each bioreactor and associated gas supply and recirculation components are configured as standalone modules that can be independently operated or seamlessly integrated into the cascade. Thus, in some embodiments, each bioreactor and gas supply and recirculation components are configured as standalone modules that can be independently operated or integrated into the series. In some embodiments, this modularity allows for the easy expansion or reduction of the system’s capacity to accommodate different production scales and operational requirements, facilitating the customization of the bioreactor cascade to specific process needs or to experiment with different microbial cultures and gaseous substrates.

[0117] In some embodiments, the system further includes a waste gas utilization module designed to capture and process exhaust gases from the cascade for secondary applications. In some embodiments, the waste gas control module efficiently separates and purifies methane, carbon dioxide, and other gaseous byproducts from the exhaust stream, rendering them suitable for use in energy generation systems, such as combined heat and power (CHP) units, or as substrates in additional fermentation processes, including but not limited to manure methanation or biohydrogen production. In some embodiments, the module includes technologies for gas cleaning, separation, and compression, tailored to meet the specifications required by downstream processes or energy systems, thereby enhancing the overallsustainability and resource efficiency of the bioreactor system, and contributing to a circular economy model by transforming waste gases into valuable resources.

[0118] In some embodiments, the system is characterized by a monitoring system for real-time analysis of gas composition, specific electricity input, and mass-transfer coefficients, to maintain safe operational parameters and achieve efficient microbial growth with a reduced carbon footprint. Thus, in some embodiments, systems include a monitoring system for real-time analysis of gas composition, specific electricity input, and mass-transfer coefficients.

[0119] In some embodiments, the optimal ratio of different gases (for example, inert gases) is determined based on the specific requirements for the cultivation of different microorganisms, including those used for the production of single-cell proteins, biofuels, biopolymers, or pharmaceutical substances.

[0120] In some embodiments, the system is implemented such that the configuration and operation of the cascade system can be adjusted to optimize an aim function selected from the group consisting of carbon footprint, gaseous substrate utilization coefficient, specific energy input, and overall system productivity. Thus, in some embodiments, the system is configured to allow optimization of an aim function selected from the group consisting of carbon footprint, gaseous substrate utilization coefficient, specific energy input, and overall system productivity.

[0121] In some embodiments, the system further includes a gas recycling module designed to ensure the explosion safety of the technological process by controlling the composition of the gas phase and maintaining safe concentrations of methane and oxygen.

[0122] In some embodiments, the bioreactor cascade system is configured to achieve high process productivity, characterized by a production rate of at least 4 kg of absolutely dry matter per hour per cubic meter of the bioreactor’s working volume. Thus, in some embodiments, the bioreactor cascade system is configured to produce at least 1 kg, at least 2 kg, at least 3 kg, at least 4 kg, at least 5 kg, or a range constructed from any of the aforementioned values, of absolutely dry matter per hour per cubic meter of working volume. In some embodiments, the bioreactor cascade system is configured to produce at least 4 kg of absolutely dry matter per hour per cubic meter of working volume.Methods for the Cultivation of Microorganisms

[0123] Disclosed herein are methods for the cultivation of microorganisms. In some embodiments, the method includes: providing a bioreactor cascade system that includes a plurality of bioreactors arranged in series, where each bioreactor is configured to receive a mixture of gaseous substrates necessary for the growth of microorganisms; providing a first bioreactor in the series with an initial supply of natural gas or an alternative gaseous carbon source (for example, a gaseous carbon source including but not limited to, biogas, methane, and associated gas), along with pressurized air, oxygen, or oxygen-enriched air; delivering a nutrient medium in gaseous form into each bioreactor via a gas supply arrangement; channeling the exhaust gas mixture from a preceding bioreactor to a succeeding bioreactor via a gas phase recirculation mechanism. In some embodiments, each bioreactor in the cascade is operatively connected to ensure the transfer of gases therethrough, and letting out exhaust gases at a predetermined stage in the cascade. In some embodiments, the predetermined stage is determined based on the operational mode but is at least a second stage.

[0124] In some embodiments, the method further includes incrementally increasing the pressure throughout the cascade to enhance solubility and mass transfer rates of gases with low solubility. In some embodiments, the method further includes enriching the gas phase with oxygen at one or more selected stages within the cascade. In some embodiments, the method further includes dynamically determining a quantity of oxygen introduced at each said stage based on the gas composition outcomes from the preceding stage(s). In some embodiments, the oxygen enrichment is independent of any additional pressure application in subsequent stages.

[0125] In some embodiments, the method further includes managing the specific energy input at each stage to maintain the required level of mass transfer efficiency. In some embodiments, the energy management negates the necessity for supplemental oxygen or additional pressure in subsequent stages of the cascade system. In some embodiments, the method further includes adjusting carbon dioxide content and / or enriching the gas mixture with pure oxygen, thereby supporting an efficient ratio of gases in the gas mixture. In some embodiments, the method further includes adjusting the composition of the gas mixture to optimize the partial pressures of oxygen and / or carbon dioxide at each stage of the cascade.

[0126] In some embodiments, the method further includes integrating a jet-stream bioreactor configuration for each stage within the cascade. In some embodiments, the bioreactors are equipped with a jet’s parameters control system. In some embodiments, the method further includes monitoring the bioreactors and dynamically adjusting the velocity, pressure, and composition of the liquid-gas jets introduced into the liquid medium. In some embodiments, the method further includes optimizing dissolution rates of methane and oxygen by continuously adapting jet parameters based on real-time analysis of microbial growth conditions, gas solubility, and mass transfer efficiency. In some embodiments, the method further includes maintaining gas mixture compositions within safe explosive limits, for example, as defined by the Gibbs-Roseboom diagram. In some embodiments, the method further includes optimizing energy consumption by adjusting jet parameters to achieve maximal substrate utilization and minimal energy expenditure.

[0127] In some embodiments, the method further includes customizing the bioreactor cascade to specific process needs or to experiment with different microbial cultures and gaseous substrates.

[0128] In some embodiments, the method further includes capturing and processing exhaust gases from the cascade for at least one secondary application. In some embodiments, the method includes separating and purifying methane, carbon dioxide, or other gaseous byproducts from the exhaust stream, and rendering the methane, carbon dioxide, or other gaseous byproducts suitable for use in energy generation systems, or as substrates in additional fermentation processes.

[0129] In some embodiments, the method further includes monitoring the series of bioreactors for real-time analysis of gas composition, specific electricity input, or mass-transfer coefficients. In some embodiments, the method further includes adjusting the configuration and operation of the cascade system to optimize an aim function selected from the group consisting of carbon footprint, gaseous substrate utilization coefficient, specific energy input, and overall system productivity.

[0130] In some embodiments, the method further includes the composition of the gas phase and maintaining safe concentrations of methane and oxygen.

[0131] In some embodiments, the method further includes producing at least 1 kg, at least 2 kg, at least 3 kg, at least 4 kg, at least 5 kg, or a range constructed from any of theaforementioned values, of absolutely dry matter per hour per cubic meter of working volume. In some embodiments, the method further includes producing at least 4 kg of absolutely dry matter per hour per cubic meter of working volume.

[0132] Additional embodiments are described in the following enumerated alternatives.

[0133] 1. A bioreactor cascade system for the cultivation of microorganisms, the system comprising: a plurality of bioreactors arranged in series, where each bioreactor is configured to receive a mixture of gaseous substrates necessary for the growth of microorganisms; the system being characterized by a gas supply arrangement that delivers components of the nutrient medium in gaseous form into each bioreactor, and a gas phase recirculation mechanism designed to channel the exhaust gas mixture from a preceding bioreactor to a succeeding bioreactor, while the first bioreactor in the series is distinctly provided with an initial supply of natural gas or an alternative gaseous carbon source, along with pressurized air, oxygen, or oxygen-enriched air; each bioreactor in the cascade is operatively connected to ensure the transfer of gases therethrough, and the system includes an outlet for the exhaust gases at a predetermined stage in the cascade, referred to as the K-step, where the number of stages is determined based on the operational mode but is not less than two.

[0134] 2. The system of alternative 1, wherein each bioreactor in the series is configured to operate at a higher pressure than the preceding bioreactor, thereby incrementally increasing the pressure throughout the cascade to enhance the solubility and mass transfer rates of gases with low solubility.

[0135] 3. The system of any one of alternatives 1 or 2, wherein the system is further adapted to enrich the gas phase with oxygen at one or more selected stages within the cascade, the quantity of oxygen introduced at each said stage being dynamically determined based on the gas composition outcomes from the preceding stage(s), such enrichment being independent of any additional pressure application in subsequent stages.

[0136] 4. The system of any one of alternatives 1-3, wherein the specific energy input at each stage is managed to maintain the required level of mass transfer efficiency, and this energy management negates the necessity for supplemental oxygen or additional pressure in subsequent stages of the cascade system.

[0137] 5. The system of any one of alternatives 1-4, further comprising a gas preparation module configured to adjust the carbon dioxide content and / or enrich the gas mixture with pure oxygen, thereby supporting an efficient ratio of gases in the gas mixture.

[0138] 6. The system of any one of alternatives 1-5, wherein the gas supply system includes a controller for adjusting the composition of the gas mixture to optimize the partial pressures of oxygen or carbon dioxide at each stage of the cascade.

[0139] 7. The system of any one of alternatives 1-6, further enhanced by integrating a jet-stream bioreactor configuration for each stage within the cascade, wherein the bioreactors are equipped with a jet’s parameters control system; this control system utilizes advanced automated monitoring and artificial intelligence, including machine learning algorithms, to dynamically adjust the velocity, pressure, and composition of the liquid-gas jets introduced into the liquid medium; the control system is specifically designed to optimize the dissolution rates of methane and oxygen by continuously adapting jet parameters based on real-time analysis of microbial growth conditions, gas solubility, and mass transfer efficiency. The control system further ensures operational safety by maintaining gas mixture compositions within safe explosive limits, as defined by the Gibbs-Roseboom diagram, and optimizes energy consumption by adjusting jet parameters to achieve maximal substrate utilization and minimal energy expenditure.

[0140] 8. The system of any one of alternatives 1-7, characterized by its modular design, wherein each bioreactor and associated gas supply and recirculation components are configured as standalone modules that can be independently operated or seamlessly integrated into the cascade; this modularity allows for the easy expansion or reduction of the system’s capacity to accommodate different production scales and operational requirements, facilitating the customization of the bioreactor cascade to specific process needs or to experiment with different microbial cultures and gaseous substrates.

[0141] 9. The system of any one of alternatives 1-8, further comprising a waste gas utilization module designed to capture and process exhaust gases from the cascade for secondary applications; this module efficiently separates and purifies methane, carbon dioxide, and other gaseous byproducts from the exhaust stream, rendering them suitable for use in energy generation systems, such as combined heat and power (CHP) units, or as substrates in additional fermentation processes, including but not limited to manure methanation orbiohydrogen production. The module includes technologies for gas cleaning, separation, and compression, tailored to meet the specifications required by downstream processes or energy systems, thereby enhancing the overall sustainability and resource efficiency of the bioreactor system and contributing to a circular economy model by transforming waste gases into valuable resources.

[0142] 10. The system of any one of alternatives 1-9, characterized by a monitoring system for real-time analysis of gas composition, specific electricity input, and mass-transfer coefficients, to maintain safe operational parameters and achieve efficient microbial growth with a reduced carbon footprint.

[0143] 11. The system of any one of alternatives 1-10, wherein the optimal ratio of different gases is determined based on the specific requirements for the cultivation of different microorganisms, including those used for the production of single-cell proteins, biofuels, biopolymers, or pharmaceutical substances.

[0144] 12. The system of any one of alternatives 1-11, implemented such that the configuration and operation of the cascade system can be adjusted to optimize an aim function selected from the group consisting of carbon footprint, gaseous substrate utilization coefficient, specific energy input, and overall system productivity.

[0145] 13. The system of any one of alternatives 1-12, further comprising a gas recycling module designed to ensure the explosion safety of the technological process by controlling the composition of the gas phase and maintaining safe concentrations of methane and oxygen.

[0146] 14. The system of any one of alternatives 1-13, wherein the bioreactor cascade system is configured to achieve high process productivity, characterized by a production rate of at least 4 kg of absolutely dry matter per hour per cubic meter of the bioreactor’s working volume.

[0147] 15. The system of any one of alternatives 1-4, wherein the alternative gaseous carbon source is biogas, methane, or associated gas.

[0148] 16. A system comprising: a plurality of bioreactors arranged in series, wherein each bioreactor is configured to receive a mixture of gaseous substrates necessary for the growth of microorganisms; a gas supply arrangement that delivers carbon and energysources in gaseous form into each bioreactor; and a gas phase recirculation mechanism that channels the exhaust gas mixture from a preceding bioreactor to a succeeding bioreactor.

[0149] 17. The system of alternative 16, wherein each bioreactor in the series is operatively connected to ensure the transfer of gases therethrough.

[0150] 18. The system of any one of alternatives 16-17, wherein a first bioreactor in the series is configured to receive an initial supply of natural gas or an alternative gaseous carbon source along with pressurized air, oxygen, or oxygen-enriched air.

[0151] 19. The system of alternative 18, wherein the alternative gaseous carbon source is biogas, methane, or associated gas.

[0152] 20. The system of any of alternatives 16-18, wherein the plurality of bioreactors arranged in series are configured to operate in a cascade.

[0153] 21. The system of alternative 20, wherein the system comprises an outlet for the exhaust gases at a predetermined stage in the cascade.

[0154] 22. The system of alternative 21, wherein the predetermined stage is determined based on the operational mode.

[0155] 23. The system of any of alternatives 20-22, wherein the cascade comprises at least two stages.

[0156] 24. The system according to any of alternatives 16-23, wherein each bioreactor in the series is configured to operate at a higher pressure than the preceding bioreactor.

[0157] 25. The system according to any of alternatives 16-24, wherein the system is further configured to enrich the gas phase with oxygen at one or more selected stages within the series.

[0158] 26. The system according to alternative 25, wherein the quantity of oxygen introduced at each said stage is dynamically determined based on the gas composition outcomes from at least one preceding stage.

[0159] 27. The system according to any one of alternatives 23-24, wherein the enrichment is independent of any additional pressure application in subsequent stages.

[0160] 28. The system according to any of alternatives 16-27, wherein the system is configured such that a specific energy input at each stage is managed to maintain the required level of mass transfer efficiency.

[0161] 29. The system according to alternative 28, wherein the system is configured such that energy management negates the necessity for supplemental oxygen or additional pressure in subsequent stages of the cascade system.

[0162] 30. The system according to any of alternatives 16-29, further comprising a gas preparation module configured to adjust the carbon dioxide content and / or enrich the gas mixture with pure oxygen, thereby supporting an efficient ratio of gases in the gas mixture.

[0163] 31. The system according to any of alternatives 16-30, wherein the gas supply arrangement includes a controller for adjusting the composition of the gas mixture to optimize the partial pressures of oxygen or carbon dioxide at each stage of the cascade.

[0164] 32. The system according to any of alternatives 16-31, wherein the system further comprises a jet-stream bioreactor configuration for each stage within the cascade.

[0165] 33. The system according to alternative 32, wherein the bioreactors are equipped with a jet parameters control system.

[0166] 34. The system according to alternative 33, wherein the jet parameters control system utilizes advanced automated monitoring and artificial intelligence to dynamically adjust the velocity, pressure, and composition of the liquid-gas jets introduced into the liquid medium.

[0167] 35. The system according to any one of alternatives 31-32, wherein the jet parameters control system is configured to optimize dissolution rates of methane and oxygen by continuously adapting jet parameters based on real-time analysis of microbial growth conditions, gas solubility, and mass transfer efficiency.

[0168] 36. The system according to any of alternatives 33-35, wherein the jet parameters control system maintains gas mixture compositions within safe explosive limits, as defined by the Gibbs-Roseboom diagram.

[0169] 37. The system according to any of alternatives 33-36, wherein the jet parameters control system is configured to optimize energy consumption by adjusting jet parameters to achieve maximal substrate utilization and minimal energy expenditure.

[0170] 38. The system according to any of alternatives 33-37, wherein each bioreactor and gas supply and recirculation component are configured as standalone modules that can be independently operated or integrated into the series.

[0171] 39. The system according to any of alternatives 16-38, further comprising a waste gas utilization module configured to capture and process exhaust gases from the cascade for secondary applications.

[0172] 40. The system of alternative 39, wherein the waste gas module separates and purifies methane, carbon dioxide, and other gaseous byproducts from the exhaust stream.

[0173] 41. The system according to any one of alternatives 16-38, further comprising a monitoring system for real-time analysis of gas composition, specific electricity input, and mass-transfer coefficients.

[0174] 42. The system according to any of alternatives 16-41, wherein the system is configured to allow optimization of an aim function selected from the group consisting of carbon footprint, gaseous substrate utilization coefficient, specific energy input, and overall system productivity.

[0175] 43. The system according any of alternatives 16-42, further comprising a gas recycling module configured to control a composition of a gas phase and to maintain safe concentrations of methane and oxygen.

[0176] 44. The system according to any of alternatives 16-43, wherein the bioreactor cascade system is configured to produce at least 4 kg of absolutely dry matter per hour per cubic meter of working volume.

[0177] 45. A method for the cultivation of microorganisms, the method comprising: providing a bioreactor cascade system comprising plurality of bioreactors arranged in series, where each bioreactor is configured to receive a mixture of gaseous substrates necessary for the growth of microorganisms; providing a first bioreactor in the series with an initial supply of natural gas or an alternative gaseous carbon source, along with pressurized air, oxygen, or oxygen-enriched air; delivering a nutrient medium in gaseous form into each bioreactor via a gas supply arrangement; channeling the exhaust gas mixture from a preceding bioreactor to a succeeding bioreactor via a gas phase recirculation mechanism, wherein each bioreactor in the cascade is operatively connected to ensure the transfer of gases therethrough, and letting out exhaust gases at a predetermined stage in the cascade, wherein the predetermined stage is determined based on the operational mode but is at least a second stage.

[0178] 46. The method according to alternative 45, wherein the method further comprises incrementally increasing the pressure throughout the cascade to enhance solubility and mass transfer rates of gases with low solubility.

[0179] 47. The method according to any one of alternatives 45-46, further comprising enriching the gas phase with oxygen at one or more selected stages within the cascade.

[0180] 48. The method according to alternative 47, further comprising dynamically determining a quantity of oxygen introduced at each said stage based on the gas composition outcomes from the preceding stage(s).

[0181] 49. The method of any one of alternatives 45-46, wherein the oxygen enrichment is independent of any additional pressure application in subsequent stages.

[0182] 50. The method of any of alternatives 45-49, further comprising managing the specific energy input at each stage to maintain the required level of mass transfer efficiency.

[0183] 51. The method of alternative 50, wherein the energy management negates the necessity for supplemental oxygen or additional pressure in subsequent stages of the cascade system.

[0184] 52. The method of any of alternatives 45-51, further comprising adjusting carbon dioxide content or enriching the gas mixture with pure oxygen, thereby supporting an efficient ratio of gases in the gas mixture.

[0185] 53. The method of any of alternatives 45-52, further comprising adjusting the composition of the gas mixture to optimize the partial pressures of oxygen or carbon dioxide at each stage of the cascade.

[0186] 54. The method of any of alternatives 45-53, further comprising integrating a jet-stream bioreactor configuration for each stage within the cascade, wherein the bioreactors are equipped with a jet’s parameters control system.

[0187] 55. The method of alternative 54, further comprising monitoring the bioreactors and dynamically adjusting the velocity, pressure, and composition of the liquid- gas jets introduced into the liquid medium.

[0188] 56. The method of any one of alternatives 54-55, further comprising optimizing dissolution rates of methane and oxygen by continuously adapting jet parametersbased on real-time analysis of microbial growth conditions, gas solubility, and mass transfer efficiency.

[0189] 57. The method of any of alternatives 54-56, further comprising maintaining gas mixture compositions within safe explosive limits.

[0190] 58. The method of any of alternatives 54-57, further comprising optimizing energy consumption by adjusting jet parameters to achieve maximal substrate utilization and minimal energy expenditure.

[0191] 59. The method according to any of alternatives 45-58, further comprising customizing the bioreactor cascade to specific process needs or to experiment with different microbial cultures and gaseous substrates.

[0192] 60. The method according to any of alternatives 45-59, further comprising capturing and processing exhaust gases from the cascade for at least one secondary application.

[0193] 61. The method according to alternative 60, wherein the method comprises separating and purifying methane, carbon dioxide, or other gaseous byproducts from the exhaust stream, and rendering the methane, carbon dioxide, or other gaseous byproducts suitable for use in energy generation systems, or as substrates in additional fermentation processes.

[0194] 62. The method according to any of alternatives 45-61, wherein the method further comprises monitoring the series of bioreactors for real-time analysis of gas composition, specific electricity input, or mass-transfer coefficients.

[0195] 63. The method according to any of alternatives 45-62, further comprising adjusting the configuration and operation of the cascade system to optimize an aim function selected from the group consisting of carbon footprint, gaseous substrate utilization coefficient, specific energy input, and overall system productivity.

[0196] 64. The method according to any of alternatives 45-63, further comprising controlling the composition of the gas phase and maintaining safe concentrations of methane and oxygen.

[0197] 65. The method of any of alternatives 45-64, further comprising producing at least 4 kg of absolutely dry matter per hour per cubic meter of working volume.

[0198] 66. The method of alternative 45, wherein the alternative gaseous carbon source is biogas, methane, or associated gas.EXAMPLES

[0199] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure. Those in the art will appreciate that many other embodiments also fall within the scope of the disclosure, as it is described herein above and in the claims. Example 1

[0200] In the following example, a bioreactor cascade system is described.

[0201] Figure 2 represents an advanced bioreactor cascade system, engineered for the cultivation of microorganisms with an integrated gas management protocol to fine-tune the environmental conditions within each bioreactor stage. The bioreactor cascade system of Figure 2 includes the following components:

[0202] Nutrition Solution Inlet (1): A nutrient-rich solution is introduced into the cascade, providing essential nutrients required for the growth and metabolism of the microorganisms.

[0203] Bioreactor Stages (I-IV): The system includes a minimum of two, and preferably seven, bioreactor stages for scalability and flexibility. Figure 2 illustrates four such stages for clarity. Each stage houses a bioreactor where the microbial culture thrives in the liquid medium. The system is designed to collect biomass from each bioreactor stage.

[0204] Main Gas Flow (6): This flow line illustrates the path of the gas mixture, which includes nitrogen, oxygen, methane, and carbon dioxide, as it moves through the system.

[0205] Gas Transfer (4.1, 4.2, 4.3): These arrows indicate the direction of the off- gas from each bioreactor stage. The off-gas is subject to purification and oxygen enrichment before being recompressed and reintroduced to the cascade. This process is dependent on the chosen management scheme and ensures the optimal gas composition for each stage.

[0206] Make-up Compressor (5): The make-up compressor unit is responsible for pressurizing additional oxygen before it enters the bioreactor stages.

[0207] Oxygen Enrichment Points (5.1, 5.2, 5.3): At these junctures, oxygen is added to the gas flow to maintain the required oxygen levels for microbial respiration.

[0208] Pressure Compressors (8): These compressors are located between each bioreactor stage, where they are tasked with increasing the pressure of the gas mixture fromthe preceding stage before it is fed into the next, ensuring that gas solubility and mass transfer rates are optimized.

[0209] Gas Preparation Modules (9.1-9.3): These units treat the off-gas to adjust the carbon dioxide concentration, ensuring that the gas mixture entering the subsequent stages is at the ideal composition to support microbial growth.

[0210] Biomass Outlet (10): Biomass produced from each stage is collected and directed out of the system for further utilization, such as downstream processing or as a product in various applications.

[0211] Figure 2 is an exemplary embodiment of some capabilities of a bioreactor cascade system to maintain a controlled, efficient environment for the cultivation of a wide variety of microorganisms. The integrated gas management system ensures that the gas composition, pressure, and biomass collection are finely tuned for each stage, resulting in a high degree of efficiency and flexibility in the microbial cultivation process. Example 2

[0212] In the following example, a cascade fermentation system was calculated according to an air scheme with an intermediate carbon dioxide utilization system.

[0213] Production technological lines based on the use of gases in the process have a low degree of use of the gas phase and as a result, the process becomes energy-intensive. A large amount of unused gases containing a high concentration of the initial gaseous raw materials was emitted.

[0214] The cascade fermentation system advantageously increased the degree of use of the gas phase and reduced specific energy costs by using a cascade circuit for switching on technological equipment.

[0215] The use of a cascade circuit can be considered on the example of a technological line for the production of single cell protein based on methane from natural gas. Natural gas and air from the atmosphere were supplied to the production process. The degree of use of a mixture of gases of one technological apparatus ranged from 50-70%, depending on the design features. When installing devices according to a parallel scheme, the utilization stage was equal to the degree of utilization of one device.

[0216] The main parameters of the processing line when installing the devices sequentially according to a cascade circuit with a working volume of 100 m3were calculated.

[0217] To ensure the safe operation of the equipment, the condition of the maximum concentrations of the components of the gas mixture were met in accordance with the Gibbs-Roseboom diagram.

[0218] For the calculation, it is assumed that the process is conducted with a limitation of the oxygen concentration in the exhaust gases of no more than 12%, while the amount of natural gas supplied is not limited. The first stage

[0219] A gas mixture in the following composition was supplied to the first stage of the technological line: Table 1 Component of the gas mixture Volume (nm3) Percentage ratio (%)ure indicated in Table 2 was spent to obtain the product, while carbon dioxide was formed as a result of the process, the volume of which is also shown in Table 2. Table 2 Component of the gas mixture Volume 3

[0221] Asa resu o e opera on o e rs s age, ex aust gas was formed, which was sent to the second stage inlet in the following composition: Oxygen: 1386 – 970 = 416 nm3 (concentration of 8%)Natural gas: 5208 – 729 = 4479 nm3 (concentration 86%)

[0222] Carbon dioxide, 321 nm3(concentration 6%) A product in the volume of 375 kg / hour was also produced, based on this, the energy costs of the second stage were determined, taking into account the energy spent on compressing the gas phase.

[0223] To ensure such performance, the oxygen sorption rate should be at least Mൌ P ൈ ^^^^ଶ ൌ 375 ∗ 3.7 ൌ 1387.5^^^^O2ൗ ℎ^^^^^^

[0224] At the operating temperatures of the plant, the equilibrium oxygen concentration in water is 6 mg / l, in which case the driving force of the sorption process can be defined as: ∆C ൌ ^^ைଶ ൈ ^^^^ െ ^^^ ൌ ^1 ^ 4^ ൈ 0,08 ൈ 28,8 െ 0,1 ൌ 11,4^^ ൌ ^^^^^^ ∗ ∆C

[0225] The formula m ൌ KLa ∗ ∆C represents the mass transfer rate of gas (oroxygen) into liquid in a fermentation system, where: M is the mass transfer rate (kg / m^3 / hour) KLa is the mass transfer coefficient (1 / hour) ∆C is the concentration difference of gas (or oxygen) between the gas-liquid interface and bulk liquid (kg / m^3)

[0226] Based on the empirical formula of the dependence of the volumetric mass transfer coefficient on the specific energy costs, the amount of energy required to ensure the process was calculated: బ.^ఱ ^^^^^^ బ.^ఱM ∗ 1000^∆ బ.^ఱM ∗ 1000^^^^^^ ^^^^ ൌ ^ ൌC^350 350∗ 2.85

[0227] was also toenergy to compress gases to operating pressure. Oxygen compression energy Ipt=259.7×288×In4+11=120.375, kJ / kg Nizt=120.3753600×1982 kg O2= 66.3, kWNef=66.30.7×0.95×100 m3=0.99, kW / m3 Methane compression energy Ipt=518×288×In4+11=240.1, kJ / kg Nizt=240.13600×3718 kg= 248, kW Nef=2480.7×0.95×100=3.72, kW / m3

[0228] The specific energy of the first stage of the cascade will be: 2.85+0.99+3.72 = 7.56 kW / m3

[0229] This indicator is brought to the specific productivity of the process: 7.56 / 3.75 = 2.02 kW / kg The second stage The used gas was supplied to the second stage of the processing line. The composition of the feed is shown in Table 3: Table 3 Component of the gas mixture Component of the gas mixture Percentage (%) Volume Volume (nm3), nal oxygen was supplied to the second stage in a volume of 935 nm3 / hour. The consumption of the gas mixture for the production of the product occurred in accordance with the costs accepted in Table 2.

[0231] As a result of the operation of the second stage, exhaust gas was formed, which was directed to the entrance of the third stage in the following composition: Oxygen: 416 + 9–5 - 970 = 381 nm3(concentration of 8%) Natural gas: 4479 – 729 = 3750 nm3(concentration 79%) Carbon dioxide: 321 + 321 = 642 nm3(concentration of 13%)

[0232] A product in the amount of 375 kg / hour was also produced, based on this, the energy costs at the second stage were determined, taking into account the energy spent on compressing the additionally supplied oxygen.

[0233] Since the partial pressure of oxygen in the composition of the gas medium did not change, the specific power consumption of the device did not differ from the first బ.^ఱ ^^^^ బ.^ఱ^^ ൈ 1000^ ௩^ ^ ^^^^^^ ^^ ൌ350ൌ^^^^ ൈ 350 ൌ 2.85^^ଷ ∙ ℎ ∙ ^^^^Energy of Oxygen compression:4^ 1^^^^௧ ൌ 259,7 ൈ 288 ൈ ^^^^1ൌ 120.375, ĸ ,Д,Ж⁄ ĸ ^120.375 ^^^^௧ൌ 3600ൈ 654ĸгO2 ൌ 21.9, ĸ^^т21.9 ^^^^^ൌ 0.7 ൈ 0.95 ൈ 100м3 ൌ 0.33,ĸ^^т⁄ м3

[0234] The specific energy of the second stage of the cascade will be: 2.85 + 0.33 = 3.18 kW / m3

[0235] This indicator is converted to the specific performance of the process: 3.18 / 3.75 = 0.85 kW / kg The third stage

[0236] The used gas was supplied to the third stage of the technological line. The composition of the supplied is given in Table 4. Table 4 Component Volume (nm3) Percentage Ratio (%) , additionaloxygen was supplied to the third stage at a rate of 925 nm3 / hour. The consumption of the gas mixture for product production was in accordance with the expenditures set in Table 2. As a result of the third stage’s operation, a spent gas was formed, which was directed to the entrance of the fourth stage with the following composition: Oxygen: 381 + 9–5 - 970 = 336 nm3 (concentration 8%) Natural Gas: 3750 – 729 = 3021 nm3 (concentration 70%)Carbon Dioxide: 642 + 321 = 963 nm3 (concentration 22%)

[0238] A product was also produced at a volume of 375 kg / hour. Based on this, the energy costs at the third stage were determined, taking into account the energy expended on compressing the additionally supplied oxygen. Since the partial pressure of oxygen in the gas mixture composition did not change, the specific consumption of electricity by the device did not differ from the previous stages బ.^ఱ ^^^^^బ.^ఱ^^ ൈ 1000^^^^^^ ^^௩ ൌ ^ ^350ൌ^^^^ ൈ 350 ൌ 2.85^^ଷ ∙ ℎ ∙ ^^^^Oxygen compression energy I_ist = 259.7 × 288 × In(4+1) / 1 = 120,375 kJ / kg N_ist = 120,375 / 3600 × 647 kg O2 = 21.6 kW N_eff = 21.6 / (0.7 × 0.95 × 100 m3) = 0.32 kW / m3

[0239] The specific energy of the third stage of the cascade will be: 2.85 + 0.32 = 3.17 kW / m3. This indicator is converted to the specific performance of the process: 3.17 / 3.75 = 0.85 kW / kg The fourth stage

[0240] The used gas was supplied to the fourth stage of the technological line. Due to the high concentration of carbon dioxide that was produced, an intermediate purification of the gas mixture was carried out, reducing the carbon dioxide to 20% by volume. The composition of the supplied is presented in Table 5. Table 5 Component Volume (nm3) Percentage Ratio (%)

[0241] To ensure the process with the necessary amount of oxygen, additional oxygen was supplied to the fourth stage at a volume of 889 nm3 / hour. The consumption of the gas mixture for product production was in accordance with the expenditures set in Table 2.

[0242] As a result of the fourth stage’s operation, a spent gas was formed, which was directed to the entrance of the fifth stage with the following composition: Oxygen: 336 + 8–9 - 970 = 255 nm3(concentration 8%) Natural Gas: 3021 – 729 = 2292 nm3(concentration 70%) Carbon Dioxide: 840 + 321 = 1161 nm3(concentration 22%)

[0243] A product was also produced at a volume of 375 kg / hour. Based on this, the energy costs at the fourth stage were determined, taking into account the energy spent on compressing the additionally supplied oxygen.

[0244] Since the partial pressure of oxygen in the gas mixture composition did not change, the specific consumption of electricity by the device did not differ from the previous stages. బ.^ఱ ^^^^ బ.^ఱ^^ ൈ 1000^^^^ ௩^ ^ ^^^ ^^ ൌ350ൌ0 ൌ 2.85^^ଷ ∙ ℎ ∙ ^^^^Energy of oxygen compression I_ist = 259.7 × 288 × In(5 / 1) = 120,375 kJ / kg N_ist = 120,375 / 3600 × 647 kg O2 = 21.6 kW N_eff = 21.6 / (0.7 × 0.95 × 100 m³) = 0.32 kW / m³ The specific energy of the fourth stage of the cascade will be: 2.85 + 0.32 = 3.17 kW / m³. This indicator is converted to the specific productivity of the process: 3.17 / 3.75 = 0.85 kW / kg The Fifth stage

[0245] The used gas was supplied to the fifth stage of the technological line. Due to the high concentration of carbon dioxide that was produced, an intermediate purification ofthe gas mixture was carried out, reducing the carbon dioxide to 20% by volume. The composition of the supplied gas is presented in Table 6. Table 6 Component of Gas Mixture Volume (nm3) Percentage Ratio (%) Ox en 225 8nal oxygen was supplied to the fifth stage at a volume of 889 nm3 / hour. The consumption of the gas mixture for product production was in accordance with the expenditures set in Table 2. As a result of the fifth stage’s operation, a spent gas was formed, which was directed to the entrance of the sixth stage with the following composition: Oxygen: 225 + 8–9 - 970 = 144 nm3(concentration 7%) Natural Gas: 2292 – 729 = 1563 nm3(concentration 62%) Carbon Dioxide: 639 + 321 = 961 nm3(concentration 31%)

[0247] A product was also produced at a volume of 375 kg / hour. Based on this, the energy costs at the fifth stage were determined, taking into account the energy spent on compressing the additionally supplied oxygen. Since the partial pressure of oxygen in the gas mixture composition did not change, the specific consumption of electricity by the device did not differ from the previous stages. బ.^ఱ ^ ^^^^^బ.^ఱ ^^^ ൈ 1000^^^^^^ ^^௩ ൌ350ൌ0 ൌ 2.85^^ଷ ∙ ℎ ∙ ^^^^Oxygen compression energy I_{ist} = 259.7 x 288 x In(5 / 1) = 120.375, kJ / kg N_{ist} = 120.375 / 3600 x 647 kg O2 = 21.6, kW N_{eff} = 21.6 / (0.7 x 0.95 x 100 m3) = 0.32, kW / m3

[0248] The specific energy of the fifth stage of the cascade will be: 2.85 + 0.32 = 3.17 kW / m3

[0249] This indicator is converted to the specific productivity of the process:3.17 / 3.75 = 0.85 kW / kg The Sixth stage The used gas was supplied to the sixth stage of the technological line. Due to the high concentration of carbon dioxide that was produced, an intermediate purification of the gas mixture was carried out, reducing the carbon dioxide to 20% by volume. The composition of the supplied gas is presented in Table 7. Table 7 Component of Gas Mixture Volume (nm3) Percentage Ratio (%)nal oxygen was supplied to the sixth stage at a volume of 889 nm3 / hour. The consumption of the gas mixture for product production was in accordance with the expenditures set in Table 2.

[0251] As a result of the sixth stage’s operation, a spent gas was formed, which was directed to the entrance of the seventh stage with the following composition: Oxygen: 144 + 8–9 - 970 = 63 nm3(concentration 4%) Natural Gas: 1563 – 729 = 834 nm3(concentration 50%) Carbon Dioxide: 432 + 321 = 753 nm3(concentration 46%)

[0252] A product was also produced at a volume of 375 kg / hour. Based on this, the energy costs at the sixth stage were determined, taking into account the energy spent on compressing the additionally supplied oxygen.

[0253] Since the partial pressure of oxygen in the composition of the gas mixture did not change, the specific electricity consumption of the device did not differ from the previous ones: బ.^ఱ ^^^^^బ.^ఱ^^ ൈ 1000^^^^^^ ^^௩ ൌ ^ ^350ൌ^^^^ ൈ 350 ൌ 2.85^^ଷ ∙ ℎ ∙ ^^^^Oxygen compression energy: 5 ^^^^௧ ൌ 259.7 ൈ 288 ൈ ^^^^ ൬1^ ൌ 120.375 kJ⁄ kg^^^^௧ൌ 3600ൌ 21.6kWmଷ

[0254] The specific energy of the second stage of the cascade will be: 2.85 ^ 0.32 ൌ 3.17kW⁄ mଷ

[0255] This indicator is converted to the specific performance of the process: 3.17 3.75ൌ 0.85kW⁄ kgThe seventh stage

[0256] The used gas was fed into the seventh stage of the technological line. Due to the high concentration of carbon dioxide produced, an intermediate purification of the gas mixture was carried out, reducing the carbon dioxide to 20% by volume.

[0257] The composition of the supplied gas is presented in Table 8. Table 8 Component of Gas Mixture Volume (nm3) Percentage Ratio (%)p y yg , nal oxygen was supplied to the seventh stage at a volume of 1080 nm3 / hour. The consumption of the gas mixture for product production was in accordance with the expenditures set in Table 2.

[0259] As a result of the seventh stage’s operation, a spent gas was formed, which was directed to the technological line for the purification of used gases with the following composition: Oxygen: 63 + 10–0 - 970 = 173 nm3(concentration 12%) Natural Gas: 834 – 729 = 105 nm3(concentration 9%)Carbon Dioxide: 753 + 321 = 1074 nm3(concentration 79%)

[0260] A product was also produced at a volume of 375 kg / hour. Based on this, the energy costs at the seventh stage were determined, taking into account the energy spent on compressing the additionally supplied oxygen. Since the partial pressure of oxygen in the composition of the gas mixture did not change, the specific consumption of electricity by the device did not differ from the previous stages. బ.^ఱ ^^^^^బ.^ఱ ^^^ ൈ 1000^^^^^^ ^^௩ ൌ ^350ൌ^^^^ ൈ 350 ൌ 2.85^^ଷ ∙ ℎ ∙ ^^^^OxygenN_ist = 120.375 / 3600 × 647 kg O2 = 21.6, kW N_eff = 21.6 / (0.7 × 0.95 × 100 m3) = 0.32, kW / m3The specific energy of the sixth stage of the cascade will be: 2.85 + 0.32 = 3.17 kW / m3This indicator is converted to the specific productivity of the process: 3.17 / 3.75 = 0.85 kW / kg

[0261] As a result of the operation of the technological line of seven stages, a high level of gas utilization and reduction in energy costs was surprisingly and unexpectedly achieved.

[0262] Information on the total expenditure is consolidated into a summary table 10 Table 10 Component Supplied to the Output from the Gas Utilization

[0263] Figure 3 visually represents various parameters through the seven stages of the cascade fermentation system.

[0264] Oxygen Supply and Consumption: indicate the volume of oxygen supplied to each stage in normal cubic meters (nm3). The oxygen levels decrease progressively as it was consumed in each stage of the fermentation process.

[0265] Natural Gas Supply: represent the volume of natural gas in each stage, also in nm3. The volume decreases through the stages due to consumption in the fermentation process.

[0266] Additional Oxygen: show the volume of additional oxygen supplied to certain stages to ensure the process has the necessary amount of oxygen for optimal fermentation. This additional oxygen is supplied when the natural supply and the oxygen remaining from the previous stages are insufficient.

[0267] Carbon Dioxide Formation and Disposal: tracks the volume of carbon dioxide formed as a byproduct of the fermentation process. After the intermediate purification steps in various stages to reduce its concentration, the final volume of carbon dioxide is lower than the initial formation.

[0268] Specific Energy Costs for Compression: indicates the specific energy costs for compressing the gases to operating pressure. These costs are likely related to the energy required to compress the additional oxygen and possibly other gases in the system.

[0269] Working Pressure: represents the working pressure in atmospheres (atm) across the stages. The pressure influences the solubility of gases in the liquid medium and is an important factor in the efficiency of the fermentation process.

[0270] Stage Indicators (Numbers 1-7): The numbers correspond to the specific stages with 1-7 representing the stages of the fermentation system. This chart summarizes the dynamic changes in gas volumes, additional oxygen requirements, carbon dioxide management, and energy costs throughout the fermentation process. It shows the efficiency of oxygen utilization, the effectiveness of carbon dioxide purification, and the overall energy expenditure for gas compression, demonstrating a successful operational control of the cascade fermentation system. Other Considerations

[0271] Conditional language used herein, such as, among others, “can,” “might,” “may,” “e.g.,” and the like, unless specifically stated otherwise, or otherwise understood withinthe context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements and / or states. Thus, such conditional language is not generally intended to imply that features, elements and / or states are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without author input or prompting, whether these features, elements and / or states are included or are to be performed in any particular embodiment. The terms “comprising,” “including,” “having,” “involving,” and the like are synonymous and are used inclusively, in an open-ended fashion, and do not exclude additional elements, features, acts, operations, and so forth. Also, the term “or” is used in its inclusive sense (and not in its exclusive sense) so that when used, for example, to connect a list of elements, the term “or” means one, some, or all of the elements in the list.

[0272] Disjunctive language such as the phrase “at least one of X, Y or Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to present that an item, term, etc., may be either X, Y or Z, or any combination thereof (such as X, Y and / or Z). Thus, such disjunctive language is not generally intended to, and should not, imply that certain embodiments require at least one of X, at least one of Y or at least one of Z to each be present.

[0273] The terms “about” or “approximate” and the like are synonymous and are used to indicate that the value modified by the term has an understood range associated with it, where the range can be ±20%, ±15%, ±10%, ±5%, or ±1%. The term “substantially” is used to indicate that a result (such as a measurement value) is close to a targeted value, where close can mean, for example, the result is within 80% of the value, within 90% of the value, within 95% of the value, or within 99% of the value.

[0274] Unless otherwise explicitly stated, articles such as “a” or “an” should generally be interpreted to include one or more described items.

[0275] While the above detailed description has shown, described, and pointed out novel features as applied to illustrative embodiments, it will be understood that various omissions, substitutions, and changes in the form and details of the devices or algorithms illustrated can be made without departing from the spirit of the disclosure. As will be recognized, certain embodiments described herein can be embodied within a form that does not provide all of the features and benefits set forth herein, as some features can be used orpracticed separately from others. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

[0276] It should be appreciated that all combinations of the foregoing concepts (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein.

[0277] The scope of the present disclosure is not intended to be limited by the specific disclosures of examples in this section or elsewhere in this specification, and may be defined by claims as presented in this section or elsewhere in this specification or as presented in the future. The language of the claims is to be interpreted broadly based on the language employed in the claims and not limited to the examples described in the present specification or during the prosecution of the application, which examples are to be construed as non- exclusive.

Claims

WHAT IS CLAIMED IS:

1. A bioreactor cascade system for the cultivation of microorganisms, the system comprising: a plurality of bioreactors arranged in series, where each bioreactor is configured to receive a mixture of gaseous substrates necessary for the growth of microorganisms; the system being characterized by a gas supply arrangement that delivers components of the nutrient medium in gaseous form into each bioreactor, and a gas phase recirculation mechanism designed to channel the exhaust gas mixture from a preceding bioreactor to a succeeding bioreactor, while the first bioreactor in the series is distinctly provided with an initial supply of natural gas or an alternative gaseous carbon source, along with pressurized air, oxygen, or oxygen-enriched air; each bioreactor in the cascade is operatively connected to ensure the transfer of gases therethrough, and the system includes an outlet for the exhaust gases at a predetermined stage in the cascade, referred to as the K-step, where the number of stages is determined based on the operational mode but is not less than two.

2. The system of claim 1, wherein each bioreactor in the series is configured to operate at a higher pressure than the preceding bioreactor, thereby incrementally increasing the pressure throughout the cascade to enhance the solubility and mass transfer rates of gases with low solubility.

3. The system of claim 1, wherein the system is further adapted to enrich the gas phase with oxygen at one or more selected stages within the cascade, the quantity of oxygen introduced at each said stage being dynamically determined based on the gas composition outcomes from the preceding stage(s), such enrichment being independent of any additional pressure application in subsequent stages.

4. The system of claim 1, wherein the specific energy input at each stage is managed to maintain the required level of mass transfer efficiency, and this energy management negates the necessity for supplemental oxygen or additional pressure in subsequent stages of the cascade system.

5. The system of claim 1, further comprising a gas preparation module configured to adjust the carbon dioxide content and / or enrich the gas mixture with pure oxygen, thereby supporting an efficient ratio of gases in the gas mixture.

6. The system of claim 1, wherein the gas supply system includes a controller for adjusting the composition of the gas mixture to optimize the partial pressures of oxygen or carbon dioxide at each stage of the cascade.

7. The system of claim 1, further enhanced by integrating a jet-stream bioreactor configuration for each stage within the cascade, wherein the bioreactors are equipped with a jet’s parameters control system; this control system utilizes advanced automated monitoring and artificial intelligence, including machine learning algorithms, to dynamically adjust the velocity, pressure, and composition of the liquid-gas jets introduced into the liquid medium; the control system is specifically designed to optimize the dissolution rates of methane and oxygen by continuously adapting jet parameters based on real-time analysis of microbial growth conditions, gas solubility, and mass transfer efficiency. The control system further ensures operational safety by maintaining gas mixture compositions within safe explosive limits, as defined by the Gibbs-Roseboom diagram, and optimizes energy consumption by adjusting jet parameters to achieve maximal substrate utilization and minimal energy expenditure.

8. The system of claim 1, characterized by its modular design, wherein each bioreactor and associated gas supply and recirculation components are configured as standalone modules that can be independently operated or seamlessly integrated into the cascade; this modularity allows for the easy expansion or reduction of the system’s capacity to accommodate different production scales and operational requirements, facilitating the customization of the bioreactor cascade to specific process needs or to experiment with different microbial cultures and gaseous substrates.

9. The system of claim 1, further comprising a waste gas utilization module designed to capture and process exhaust gases from the cascade for secondary applications; this module efficiently separates and purifies methane, carbon dioxide, and other gaseous byproducts from the exhaust stream, rendering them suitable for use in energy generation systems, such as combined heat and power (CHP) units, or as substrates in additional fermentation processes, including but not limited to manure methanation or biohydrogen production. The module includes technologies for gas cleaning, separation, and compression, tailored to meet the specifications required by downstream processes or energy systems, thereby enhancing the overall sustainability and resource efficiency of the bioreactor systemand contributing to a circular economy model by transforming waste gases into valuable resources.

10. The system of claim 1, characterized by a monitoring system for real-time analysis of gas composition, specific electricity input, and mass-transfer coefficients, to maintain safe operational parameters and achieve efficient microbial growth with a reduced carbon footprint.

11. The system of claim 1, wherein the optimal ratio of different gases is determined based on the specific requirements for the cultivation of different microorganisms, including those used for the production of single-cell proteins, biofuels, biopolymers, or pharmaceutical substances.

12. The system of claim 1, implemented such that the configuration and operation of the cascade system can be adjusted to optimize an aim function selected from the group consisting of carbon footprint, gaseous substrate utilization coefficient, specific energy input, and overall system productivity.

13. The system of claim 1, further comprising a gas recycling module designed to ensure the explosion safety of the technological process by controlling the composition of the gas phase and maintaining safe concentrations of methane and oxygen.

14. The system of claim 1, wherein the bioreactor cascade system is configured to achieve high process productivity, characterized by a production rate of at least 4 kg of absolutely dry matter per hour per cubic meter of the bioreactor’s working volume.

15. The system of claim 1, wherein the alternative gaseous carbon source is biogas, methane, or associated gas.

16. A system comprising: a plurality of bioreactors arranged in series, wherein each bioreactor is configured to receive a mixture of gaseous substrates necessary for the growth of microorganisms; a gas supply arrangement that delivers carbon and energy sources in gaseous form into each bioreactor; and a gas phase recirculation mechanism that channels the exhaust gas mixture from a preceding bioreactor to a succeeding bioreactor.

17. The system of claim 16, wherein each bioreactor in the series is operatively connected to ensure the transfer of gases therethrough.

18. The system of claim 16, wherein a first bioreactor in the series is configured to receive an initial supply of natural gas or an alternative gaseous carbon source along with pressurized air, oxygen, or oxygen-enriched air.

19. The system of claim 18, wherein the alternative gaseous carbon source is biogas, methane, or associated gas.

20. The system of claim 16, wherein the plurality of bioreactors arranged in series are configured to operate in a cascade.

21. The system of claim 20, wherein the system comprises an outlet for the exhaust gases at a predetermined stage in the cascade.

22. The system of claim 21, wherein the predetermined stage is determined based on the operational mode.

23. The system of claim 20, wherein the cascade comprises at least two stages.

24. The system of claim 16, wherein each bioreactor in the series is configured to operate at a higher pressure than the preceding bioreactor.

25. The system of claim 16, wherein the system is further configured to enrich the gas phase with oxygen at one or more selected stages within the series.

26. The system of claim 25, wherein the quantity of oxygen introduced at each said stage is dynamically determined based on the gas composition outcomes from at least one preceding stage.

27. The system of claim 23, wherein the enrichment is independent of any additional pressure application in subsequent stages.

28. The system of claim 16, wherein the system is configured such that a specific energy input at each stage is managed to maintain the required level of mass transfer efficiency.

29. The system of claim 28, wherein the system is configured such that energy management negates the necessity for supplemental oxygen or additional pressure in subsequent stages of the cascade system.

30. The system of claim 16, further comprising a gas preparation module configured to adjust the carbon dioxide content and / or enrich the gas mixture with pure oxygen, thereby supporting an efficient ratio of gases in the gas mixture.

31. The system of claim 16, wherein the gas supply arrangement includes a controller for adjusting the composition of the gas mixture to optimize the partial pressures of oxygen or carbon dioxide at each stage of the cascade.

32. The system of claim 16, wherein the system further comprises a jet-stream bioreactor configuration for each stage within the cascade.

33. The system of claim 32, wherein the bioreactors are equipped with a jet parameters control system.

34. The system of claim 33, wherein the jet parameters control system utilizes advanced automated monitoring and artificial intelligence to dynamically adjust the velocity, pressure, and composition of the liquid-gas jets introduced into the liquid medium.

35. The system of claim 33, wherein the jet parameters control system is configured to optimize dissolution rates of methane and oxygen by continuously adapting jet parameters based on real-time analysis of microbial growth conditions, gas solubility, and mass transfer efficiency.

36. The system of claim 33, wherein the jet parameters control system maintains gas mixture compositions within safe explosive limits, as defined by the Gibbs-Roseboom diagram.

37. The system of claim 33, wherein the jet parameters control system is configured to optimize energy consumption by adjusting jet parameters to achieve maximal substrate utilization and minimal energy expenditure.

38. The system of claim 33, wherein each bioreactor and gas supply and recirculation component are configured as standalone modules that can be independently operated or integrated into the series.

39. The system of claim 16, further comprising a waste gas utilization module configured to capture and process exhaust gases from the cascade for secondary applications.

40. The system of claim 39, wherein the waste gas module separates and purifies methane, carbon dioxide, and other gaseous byproducts from the exhaust stream.

41. The system of claim 16, further comprising a monitoring system for real-time analysis of gas composition, specific electricity input, and mass-transfer coefficients.

42. The system of claim 16, wherein the system is configured to allow optimization of an aim function selected from the group consisting of carbon footprint, gaseous substrate utilization coefficient, specific energy input, and overall system productivity.

43. The system of claim 16, further comprising a gas recycling module configured to control a composition of a gas phase and to maintain safe concentrations of methane and oxygen.

44. The system of claim 16, wherein the bioreactor cascade system is configured to produce at least 4 kg of absolutely dry matter per hour per cubic meter of working volume.

45. A method for the cultivation of microorganisms, the method comprising: providing a bioreactor cascade system comprising plurality of bioreactors arranged in series, where each bioreactor is configured to receive a mixture of gaseous substrates necessary for the growth of microorganisms; providing a first bioreactor in the series with an initial supply of natural gas or an alternative gaseous carbon source, along with pressurized air, oxygen, or oxygen- enriched air; delivering a nutrient medium in gaseous form into each bioreactor via a gas supply arrangement; channeling the exhaust gas mixture from a preceding bioreactor to a succeeding bioreactor via a gas phase recirculation mechanism, wherein each bioreactor in the cascade is operatively connected to ensure the transfer of gases therethrough, and letting out exhaust gases at a predetermined stage in the cascade, wherein the predetermined stage is determined based on the operational mode but is at least a second stage.

46. The method of claim 45, wherein the method further comprises incrementally increasing the pressure throughout the cascade to enhance solubility and mass transfer rates of gases with low solubility.

47. The method of claim 45, further comprising enriching the gas phase with oxygen at one or more selected stages within the cascade.

48. The method of claim 47, further comprising dynamically determining a quantity of oxygen introduced at each said stage based on the gas composition outcomes from the preceding stage(s).

49. The method of claim 45, wherein the oxygen enrichment is independent of any additional pressure application in subsequent stages.

50. The method of claim 45, further comprising managing the specific energy input at each stage to maintain the required level of mass transfer efficiency.

51. The method of claim 50, wherein the energy management negates the necessity for supplemental oxygen or additional pressure in subsequent stages of the cascade system.

52. The method of claim 45, further comprising adjusting carbon dioxide content or enriching the gas mixture with pure oxygen, thereby supporting an efficient ratio of gases in the gas mixture.

53. The method of claim 45, further comprising adjusting the composition of the gas mixture to optimize the partial pressures of oxygen or carbon dioxide at each stage of the cascade.

54. The method of claim 45, further comprising integrating a jet-stream bioreactor configuration for each stage within the cascade, wherein the bioreactors are equipped with a jet’s parameters control system.

55. The method of claim 54, further comprising monitoring the bioreactors and dynamically adjusting the velocity, pressure, and composition of the liquid-gas jets introduced into the liquid medium.

56. The method of claim 54, further comprising optimizing dissolution rates of methane and oxygen by continuously adapting jet parameters based on real-time analysis of microbial growth conditions, gas solubility, and mass transfer efficiency.

57. The method of claim 54, further comprising maintaining gas mixture compositions within safe explosive limits.

58. The method of claim 54, further comprising optimizing energy consumption by adjusting jet parameters to achieve maximal substrate utilization and minimal energy expenditure.

59. The method of claim 45, further comprising customizing the bioreactor cascade to specific process needs or to experiment with different microbial cultures and gaseous substrates.

60. The method of claim 45, further comprising capturing and processing exhaust gases from the cascade for at least one secondary application.

61. The method of claim 60, wherein the method comprises separating and purifying methane, carbon dioxide, or other gaseous byproducts from the exhaust stream, and rendering the methane, carbon dioxide, or other gaseous byproducts suitable for use in energy generation systems, or as substrates in additional fermentation processes.

62. The method of claim 45, wherein the method further comprises monitoring the series of bioreactors for real-time analysis of gas composition, specific electricity input, or mass-transfer coefficients.

63. The method of claim 45, further comprising adjusting the configuration and operation of the cascade system to optimize an aim function selected from the group consisting of carbon footprint, gaseous substrate utilization coefficient, specific energy input, and overall system productivity.

64. The method of claim 45, further comprising controlling the composition of the gas phase and maintaining safe concentrations of methane and oxygen.

65. The method of claim 45, further comprising producing at least 4 kg of absolutely dry matter per hour per cubic meter of working volume.

66. The method of claim 45, wherein the alternative gaseous carbon source is biogas, methane, or associated gas.

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