Method and apparatus for managing aerobic gas fermentation

Abstract

A method and apparatus for aerobic gas fermentation by C1-fixing microorganisms is disclosed. The present disclosure relates to a method and apparatus for increased production of microbial biomass and / or other products. In particular, the method maintains the ratio of the gaseous substrates in a unique range and provides for oxygen control in the headspace of a bioreactor.

Description

METHOD AND APPARATUS FOR MANAGING AEROBTC GAS FERMENTATIONCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 717,408, filed November 7, 2024, and of U.S. Provisional Patent Application No. 63 / 790,557, filed April 17, 2025, the entirety of both of which are incorporated herein by reference.FIELD

[0002] The present disclosure generally relates to a method and apparatus for aerobic gas fermentation. In particular, the present disclosure relates to a method and apparatus for increased production of microbial biomass and / or other products, and safe operation of aerobic fermentation.BACKGROUND

[0003] The following discussion is merely provided to aid the reader in understanding the disclosure and is not admitted to describe or constitute prior art thereto.

[0004] Climate change has created an urgent demand for the development of sustainable food from renewable resources. Further, there is a need to reduce the amount of carbon dioxide and other greenhouse gas (GHG) emissions in the atmosphere, as well as to reduce water consumption and energy consumption based upon the utilization of coal, oil, and natural gas in food production systems. Biotechnology and biomanufacturing harnesses the power of biology to create new services and products such as protein. There is a world-wide need to advance the science and engineering of biotechnology and biomanufacturing while also reducing obstacles for commercialization so that innovative technology and products can reach markets faster. Microbial gas fermentation is such a biotechnology and biomanufacturing system that may be used for the biological fixation of gases to produce protein such as single-cell protein (SCP). Microbial gas fermentation is also an example of a biotechnology and biomanufacturing system that further achieves societal climate goals while improving food security, sustainability, and reliable food chains. In particular, Cl -fixing strains have been demonstrated to convert gases containing CO2, CH3OH, CO, and / or H2 into products, including nutritive compositions as food and feed ingredients via microbial gas fermentation. Some Cl -fixing strains grow under anaerobic conditions while others grow under aerobic conditions.

[0005] During aerobic gas fermentation, unreacted oxygen separates from the fermentation broth into the headspace of the bioreactor. The unreacted oxygen mixes with other unreacted gases, e.g.,effluent gases, in the headspace of the bioreactor. Any unreacted oxygen in the gaseous mixture in the headspace of the bioreactor and effluent gases may result in flammable mixtures specifically when flammable gases (e.g., hydrogen), are used or produced in the aerobic gas fermentation. The risk of flammability and explosion is a concern for safe operation of such aerobic gas fermentation processes. Also, for aerobic fermentation it is generally preferable to operate at the highest possible oxygen concentration to maximise productivity.

[0006] There is accordingly an ongoing and unmet global need for an improved method and apparatus for increased production of microbial biomass and / or other products and safe operation of aerobic fermentation.SUMMARY OF THE INVENTION

[0007] The inventors have found an improved and safe method and apparatus for increased production of microbial biomass and / or other products by aerobic fermentation. In an embodiment, a method for aerobic gas fermentation is disclosed. The method comprising providing to a bioreactor comprising a liquid nutrient medium comprising a culture of a microorganism, a gaseous substrate comprising a first gaseous stream comprising O2 from an O2 source; a second gaseous stream comprising H2 from a H2 source; a third gaseous stream comprising CO2 from a CO2 source; feeding the second and third gaseous streams to the bioreactor at a flow rate to provide the H2 and CO2 to the bioreactor at a H2: CO2 molar ratio within a predetermined molar ratio range to attain a predetermined value of an uptake molar ratio of H2: CO2 by the microorganism; feeding the first gaseous stream to the bioreactor to provide O2 at a concentration necessary for the microorganism to uptake and ferment with the H2 and CO2 to produce microbial biomass and / or at least one chemical product and an outlet gas stream comprising O2; measuring oxygen concentration in an outlet gas stream, or an outlet gas sample stream or a headspace above the liquid nutrient medium; transmitting the measured oxygen concentration to a control system which compares the measured oxygen concentration to a predetermined value based on the limiting oxygen concentration (LOC) of hydrogen; and if the oxygen concentration is above or below the predetermined value reducing or increasing the flow rate of the first gaseous stream.

[0008] In an embodiment, the method includes introducing a diluent selected from H2, CO2, N2, Ar, or any combination thereof into the bioreactor headspace to dilute the concentration of oxygen in the headspace and maintain it below the predetermined value.

[0009] In an embodiment, the method includes compressing the outlet gas stream which comprises O2, H2 and CO2 to generate a compressed outlet gas stream and recycling at least a portion of the compressed outlet gas stream to combine with the second or third gaseous stream or directly into the liquid nutrient medium in the bioreactor.

[0010] In an embodiment, the method includes recycling a portion of the liquid nutrient medium from the bottom of the bioreactor to the top of the bioreactor headspace wherein the portion of the liquid nutrient medium is introduced into the bioreactor headspace by at least one spray nozzle.

[0011] In an embodiment, the method includes wherein the feed rate of the hydrogen in second gaseous stream comprising H2 is from about 5% to about 10% greater than the consumption rate of H2 by the microorganism.

[0012] In an embodiment, a method for aerobic gas fermentation is disclosed, which comprises providing to a bioreactor comprising a liquid nutrient medium comprising a culture of a microorganism, a gaseous substrate comprising a first gaseous stream comprising O2 from an O2 source; a second gaseous stream comprising CH4 from a CH4 source; fermenting the CH4 and O2 to produce microbial biomass and an outlet gas stream comprising O2; feeding the CH4 at a flow rate to maintain the O2 concentration in the headspace at a predetermined setpoint below the limiting oxygen concentration (LOC) for CH4 ; measuring oxygen concentration in an outlet gas stream, or an outlet gas sample stream or a bioreactor headspace above the liquid nutrient medium; transmitting the measured oxygen concentration to a control system which compares the measured oxygen concentration to the setpoint; and if the oxygen concentration is above the setpoint, reducing the flow of the first gaseous stream to the bioreactor.

[0013] In an embodiment, an apparatus for aerobic gas fermentation is disclosed. The apparatus comprising a bioreactor vessel comprising an exterior casing having a top and a bottom, the bioreactor vessel containing a liquid nutrient medium comprising a culture, a headspace above the liquid; the bioreactor vessel having at least one gas comprising O2 inlet located on the exterior casing and below the top surface of the liquid nutrient medium and in fluid communication with a gas comprising O2 supply conduit in fluid communication with a gas comprising O2 supply unit, the gas comprising O2 supply conduit further comprising a gas comprising O2 flow control valve; the bioreactor vessel having at least one gaseous substrate inlet located on the exterior casing and below the top surface of the liquid nutrient medium and in fluid communication with at least one gaseous substrate supply conduit, the gaseous substrate supply conduit in fluid communicationwith a gaseous substrate supply unit, the conduit further comprising a gaseous substrate flow control valve; an outlet gas outlet located approximate to the top of the bioreactor vessel, and in fluid communication with at least one outlet gas conduit, an outlet gas sample outlet located approximate to the top of the bioreactor vessel and in fluid communication with at least one outlet gas sample conduit, at least one oxygen sensor located on the at least one outlet gas conduit or at least one oxygen sensor located the at least one outlet gas sample conduit and optionally at least one oxygen sensor located within the headspace; and an O2 control system in electrical communication with the at least one oxygen sensor located in the outlet gas conduit, and / or the outlet gas sample conduit and with the optional at least one oxygen sensor located in the headspace of the bioreactor wherein the compressed outlet gas conduit is in fluid communication with a liquid ring compressor in fluid communication with a compressed outlet gas conduit, wherein the compressed outlet gas conduit is in fluid communication with the gaseous substrate supply conduit or with an inlet on the bioreactor vessel located below the top of the liquid nutrient medium.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following detailed description is merely exemplary in nature and is not intended to limit the various embodiments or the application and uses thereof. Furthermore, there is no intention to be bound by any theory presented in the preceding background or the following detailed description. The figures have been simplified by the deletion of a large number of apparatuses customarily employed in a process of this nature which are not specifically required to illustrate the performance of the disclosure. Furthermore, the illustration of the process of this disclosure in the embodiment of a specific drawing is not intended to limit the disclosure to specific embodiments. Some embodiments may be described by reference to the process configuration shown in the figures, which relate to both apparatus and methods to carry out the disclosure. Any reference to method includes reference to an apparatus unit or equipment that is suitable to carry out the step, and vice versa.

[0015] Fig.l shows a high level diagram of an embodiment of the disclosure.

[0016] Fig. 2 is a plot that illustrates the biomass production over time, as in the Example.

[0017] Fig. 3 is a plot depicting the headspace O2 smooth controller response necessary for maintaining the desired setpoint (SP), and further shows how the controller responded when the O2 concentration briefly went over the O2 SP. The plots in Fig. 3 present the headspace O2 controller's response to changes in O2 concentration over time as in the Example.

[0018] Fig. 4 is a top-down section view of a reactor and a distribution of an array of shower heads within the headspace of the reactor.DETAILED DESCRIPTION

[0019] The inventors have found a safe and efficient method and apparatus for increased production of microbial biomass and / or other products and safe operation of an aerobic fermentation method. The disclosed method and apparatus adjust the flow of gaseous substrate into the bioreactor vessel and / or shut off the flow of gas comprising O2, based on a measured value of molar ratio of gaseous substrate fed to the bioreactor vessel and measured concentration of O2 in the bioreactor headspace.Microorganisms for use in the disclosed method

[0020] In an embodiment, the microorganism of the disclosure is an aerobic bacterium. In one embodiment, the microorganism of the disclosure comprises aerobic hydrogen bacteria. In an embodiment, the aerobic bacteria comprising at least one disrupted gene.

[0021] A number of aerobic bacteria are known to be capable of carrying out fermentation for the disclosed method. Examples of such bacteria that are suitable for use in the disclosure include bacteria of the genus Cupriavidus and Ralstonia. In some embodiments, the aerobic bacteria is Cupriavidus necator (previously known as Alcaligenes eutrophus, Ralstonia eutropha, Wautersia eutropha or Hydrogenomonas eutropha). In some embodiments, the aerobic bacteria is Cupriavidus alkaliphilus . In some embodiments, the aerobic bacteria is Cupriavidus basilensis. In some embodiments, the aerobic bacteria is Cupriavidus campinensis. In some embodiments, the aerobic bacteria is Cupriavidus gilardii . In some embodiments, the aerobic bacteria is Cupriavidus laharis. In some embodiments, the aerobic bacteria is Cupriavidus metallidurans . In some embodiments, the aerobic bacteria is Cupriavidus nantongensis. In some embodiments, the aerobic bacteria is Cupriavidus numazuensis . In some embodiments, the aerobic bacteria is Cupriavidus oxalaticus. In some embodiments, the aerobic bacteria is Cupriavidus pampae. In some embodiments, the aerobic bacteria is Cupriavidus pauculus. In some embodiments, the aerobic bacteria is Cupriavidu pinatubonensis. In some embodiments, the aerobic bacteria is Cupriavidus plantarum. In some embodiments, the aerobic bacteria is Cupriavidus respiraculi. In some embodiments, the aerobic bacteria is Cupriavidus taiwanensis . In some embodiments, the aerobic bacteria is Cupriavidus yeoncheonensis .

[0022] In some embodiments, the strain is Cupriavidus necator DSM 428, DSM 531 , or DSM541 , or any derivatives thereof. In another embodiment, the strain is Cupriavidus necator DSM 34774.

[0023] In some embodiments, the aerobic bacteria comprises one or more exogenous nucleic acid molecules encoding a naturally occurring polypeptide, wherein the polypeptide is ribulose bisphosphate carboxylase, acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydratase, butyryl-CoA dehydrogenase, butanol dehydrogenase, electron-transferring flavoprotein large subunit, 3-hydroxybutyryl-CoA dehydrogenase, bifunctional acetaldehyde-CoA / alcohol dehydrogenase, acetaldehyde dehydrogenase, aldehyde decarbonylase, acyl-ACP reductase, L- 1,2-propanediol oxidoreductase, acyltransferase, 3-oxoacyl-ACP synthase, 3 -hydroxybutyryl - CoA epimerase / delta(3)-cis-delta(2)-trans-enoyl-CoA isomerase / enoyl-CoA hydratase / 3- hydroxyacyl-CoA dehydrogenase, short chain dehydrogenase, trans-2-enoyl-CoA reductase, or any combination thereof.

[0024] In an embodiment, the fermentation broth comprises the feed streams in combination with the aerobic microorganism in the bioreactor. In some embodiments, the feed streams, e.g., a carbon source or hydrogen feed streams, and an oxygen-containing gas feed stream, react with the microorganism in the bioreactor to at least partially form the fermentation broth (which may also include other products, byproducts, and other media fed to the bioreactor). The unreacted oxygen, or the oxygen that is not consumed by the microorganism, exists as both dissolved oxygen and gaseous oxygen in a dispersed gaseous phase within the fermentation broth. The same holds true for the other gases that are soluble. The dispersed gaseous phase, containing the unreacted components, e.g., oxygen, nitrogen, hydrogen, carbon dioxide and / or water vapor, rises to the headspace of the bioreactor.

[0025] In one embodiment, the gaseous substrate includes CO2 as a carbon source. In one embodiment, the gaseous substrate includes a mixture of gases, comprising H2 and CO2. In one embodiment, the gaseous substrate includes CPU as a carbon source.

[0026] In some embodiments, the gas fermentation product is selected from an alcohol, an acid, a diacid, an alkene, a terpene, an isoprene, and alkyne. In some embodiments, the method and microorganism disclosed herein are for the production of ethylene. In an embodiment, the method and microorganism disclosed herein are for the production of a gas fermentation product.

[0027] In one embodiment, the aerobic bacteria may produce a product such as acetone, isopropanol, 3-hydroxyisovaleryl-CoA, 3-hydroxyisovalerate, isobutylene, isopentenylpyrophosphate, dimethylallyl pyrophosphate, isoprene, famesene, 3 -hydroxybutyryl -CoA, crotonyl-CoA, 3-hydroxybutyrate, 3 -hydroxybutyrylaldehyde, 1,3 -butanediol, 2- hydroxyisobutyryl-CoA, 2-hydroxyisobutyrate, butyryl-CoA, butyrate, butanol, caproate, hexanol, octanoate, octanol, 1,3 -hexanediol, 2-buten-l-ol, isovaleryl-CoA, isovalerate, isoamyl alcohol, methacrolein, methyl-methacrylate, or any combination thereof.

[0028] In another embodiment, the bacteria of the disclosure may produce ethylene, ethanol, propane, acetate, 1 -butanol, butyrate, 2,3 -butanediol, lactate, butene, butadiene, methyl ethyl ketone (2-butanone), acetone, isopropanol, a lipid, 3-hydroxypropionate (3 -HP), a terpene, isoprene, a fatty acid, 2-butanol, 1,2-propanediol, 1-propanol, 1-hexanol, 1 -octanol, a fatty alcohol, chori smate-derived products, 3-hydroxybutyrate, 1,3 -butanediol, 2-hydroxyisobutyrate or 2- hydroxyisobutyric acid, isobutylene, adipic acid, keto-adipic acid, l,3hexanediol, 3-methyl-2- butanol, 2-buten-l-ol, isovalerate, isoamyl alcohol, and monoethylene glycol, or any combination thereof. At least some of the products may be further converted to produce at least one component of diesel, jet fuel, sustainable aviation fuel (SAP) and / or gasoline. In certain embodiments, ethylene may be catalytically converted into another product, article, or any combination thereof. In certain embodiments, microbial biomass itself is a fermentation product. Additionally, the microbial biomass may be further processed to produce a single cell protein (SCP) by any method or combination of methods known in the art. In addition to one or more target chemical products, the microorganism of the disclosure may also produce pyruvate, acetate, ethanol, 2,3-butanediol succinate, alpha-ketoglutarate, 3-hydroxybutyrate, and / or lactate. In another embodiment, the microorganism and method of the disclosure improve the production of products, proteins, microbial biomass, or any combination thereof.Substrate

[0029] “ Substrate” refers to a carbon and / or energy source for the microorganism of the disclosure. Often, the substrate is gaseous and comprises a C l -carbon source, for example, CO, CO2, CH3OH, CH2O2 and / or CH4. The substrate may further comprise an energy source such as H2.

[0030] In some embodiments, the substrate comprises CO2 and an energy source. In some embodiments, the substrate comprises CO2 and any suitable energy source. In another embodiment, the substrate comprises CO2 and H2. In yet another embodiment, the gaseous substrate comprises CH4 The amount of H2 present in the gaseous substrate varies from about 1vol. % to about 100 vol. %. For methane fermentation no or very little H2 is required. For CO2 fermentation with Cupriavidus necator greater amounts of H2 are required for the fermentation.

[0031] In an embodiment, the gas comprising oxygen may be, air i.e., 20 vol. % O2. In one embodiment, the gas comprising oxygen can be an oxy gen-enriched source, e.g., oxygen-enriched air or pure oxygen. Thus, in an embodiment, the gas comprising oxygen may comprise greater than 20.0 vol. %, greater than 40.0 vol. %, greater than 60.0 vol. %, greater than 80.0 vol. %, or greater than 90.0 vol. % or about 100 vol. % O2.

[0032] The gaseous substrate may also comprise CO2. For example, the gaseous substrate may comprise about 1 vol % to about 100 vol % CO2.

[0033] The gaseous substrate and / or Cl -carbon source may be a waste gas or an off gas obtained as a byproduct of an industrial process or from some other source, such as from automobile exhaust fumes or industrial flue gas. In certain embodiments, the industrial process is selected from the group consisting of non-ferrous products manufacturing, petroleum refining, coal gasification, electric power production, carbon black production, ammonia production, methanol production, and coke manufacturing. In these embodiments, the gaseous substrate and / or Cl -carbon source may be captured from the industrial process before it is emitted into the atmosphere, using any convenient method.

[0034] In another embodiment, the syngas may be obtained from the gasification of municipal solid waste or industrial solid waste. In an embodiment, when a syngas is used as a gaseous substrate, it is separated into various gases including H2, CO and / or CO2 which are used as individual feed gas for the bioreactor.

[0035] The terms “feedstock” when used in the context of the stream flowing into a gas fermentation bioreactor (i.e., gas fermenter) or “gas fermentation feedstock” should be understood to encompass any material (solid, liquid, or gas) or stream that can provide a substrate and / or Cl- carbon source to a gas fermenter or bioreactor either directly or after processing of the feedstock.

[0036] The term “waste gas” or “waste gas stream” may be used to refer to any gas stream that is either emitted directly, flared with no additional value capture, or combusted for energy recovery purposes.

[0037] The terms “synthesis gas” or “syngas” refers to a gaseous mixture that contains at least one carbon source, such as carbon monoxide (CO), carbon dioxide (CO2), or any combination thereof,and, optionally, hydrogen (H2) that can used as a feedstock for the disclosed gas fermentation processes and can be produced from a wide range of carbonaceous material, both solid and liquid.

[0038] The substrate and / or Cl -carbon source may be a waste gas obtained as a byproduct of an industrial process or from another source, such as biogas, landfill gas, direct air capture, or from electrolysis. The substrate and / or Cl -carbon source may be syngas generated by pyrolysis, torrefaction, or gasification.

[0039] In certain embodiments, the industrial process is selected from paper and pulp manufacturing, carbohydrate fermentation, cement making, aerobic digestion, anaerobic digestion, natural gas extraction, cellulosic fermentation, oil extraction, geological reservoirs, gas from fossil resources such as natural gas coal and oil, or any combination thereof. Examples of specific processing steps within an industrial process include catalyst regeneration, fluid catalyst cracking, and catalyst regeneration. Air separation and direct air capture are other suitable industrial processes. Specific examples in steel and ferroalloy manufacturing include blast furnace gas, basic oxygen furnace gas, coke oven gas, direct reduction of iron furnace top-gas, and residual gas from smelting iron. In these embodiments, the substrate and / or Cl -carbon source may be captured from the industrial process before it is emitted into the atmosphere, using any known method.

[0040] The substrate and / or Cl -carbon source may be synthesis gas known as syngas, which may be obtained from reforming, partial oxidation, or gasification processes. Examples of gasification processes include gasification of coal, gasification of refinery residues, gasification of petroleum coke, gasification of lignocellulosic material, gasification of waste wood, gasification of black liquor, gasification of municipal solid waste, gasification of municipal liquid waste, gasification of industrial solid waste, gasification of industrial liquid waste, gasification of refuse derived fuel, gasification of sewerage, gasification of sewerage sludge, gasification of sludge from wastewater treatment, gasification of biogas. Examples of reforming processes include, steam methane reforming, steam naphtha reforming, reforming of natural gas, reforming of biogas, reforming of landfill gas, naphtha reforming, and dry methane reforming. Examples of partial oxidation processes include thermal and catalytic partial oxidation processes, catalytic partial oxidation of natural gas, partial oxidation of hydrocarbons. Examples of municipal solid waste include tires, plastics, fibers, such as in shoes, apparel, and textiles. Municipal solid waste may be simply landfill-type waste. The municipal solid waste may be sorted or unsorted. Examples of biomassmay include lignocellulosic material and may also include microbial biomass. Lignocellulosic material may include agriculture waste and forest waste.

[0041] The substrate and / or Cl -carbon source may be a gas stream comprising methane. Such a gas comprising methane may be obtained from fossil methane emission such as during fracking, wastewater treatment, livestock, agriculture, anaerobic digestion of biomass, and municipal solid waste landfills. It is also envisioned that the methane may be burned to produce electricity or heat, and the Cl byproducts may be used as the substrate or carbon source.

[0042] “Microbial biomass” refers to biological material comprising microorganism cells. For example, microbial biomass may comprise or consist of a pure or substantially pure culture of a bacterium, archaea, virus, or fungus. When initially separated from a fermentation broth, microbial biomass generally contains a large amount of water. This water may be removed or reduced by drying or processing the microbial biomass.

[0043] The microbial biomass may comprise any of the components listed in this application but is not limited to the disclosures herein. Accordingly, the values may refer to amounts of each component per amount of wet (i.e., non-dried) microbial biomass. Herein, the composition of the microbial biomass is described in terms of weight of a component per weight of wet (i.e., nondried) microbial biomass. Of course, it is also possible to calculate the composition of the microbial biomass in terms of weight of a component per weight of dry microbial biomass.

[0044] The microbial biomass generally contains a large fraction of protein, such as more than 50% (50 g protein / 100 g biomass), more than 60% (60 g protein / 100 g biomass), more than 70% (70 g protein / 100 g biomass), or more than 80% (80 g protein / 100 g biomass) protein by weight. In an embodiment, the microbial biomass comprises at least 72% (72 g protein / 100 g biomass) protein by weight. The protein fraction comprises amino acids, including aspartic acid, alanine, arginine, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and / or valine. In particular, the microbial biomass may comprise more than 10 mg methionine / g biomass, more than 15 mg methionine / g biomass, more than 20 mg methionine / g biomass, or more than 25 mg methionine / g biomass. In an embodiment, the microbial biomass comprises at least 17.6 mg methionine / g biomass.

[0045] The microbial biomass may contain a number of vitamins, including vitamins A (retinol), C, Bl (thiamine), B2 (riboflavin), B3 (niacin), B5 (pantothenic acid), and / or B6 (pyridoxine).

[0046] The microbial biomass may contain relatively small amounts of carbohydrates and fats. For example, the microbial biomass may comprise less than 15% (15 g carbohydrate / 100 g biomass), less than 10% (10 g carbohydrate / 100 g biomass), or less than 5% (5 g carbohydrate / 100 g biomass) of carbohydrate by weight. For example, the microbial biomass may comprise less than 10% (10 g fat / 100 g biomass), or less than 5% (5 g fat / 100 g biomass), less than 2% (2 g fat / 100 g biomass), or less than 1% (1 g fat / 100 g biomass) of fat by weight.

[0047] “Selectivity” refers to the ratio of the production of a target product to the production of all fermentation products produced by a microorganism. The microorganism of the disclosure may be engineered to produce products at a certain selectivity or at a minimum selectivity. At least one of the one or more fermentation products may be biomass produced by the culture. At least a portion of the microbial biomass may be converted to a single cell protein (SCP). At least a portion of the single cell protein may be utilized as a component of animal feed.

[0048] A “single cell protein” (SCP) refers to a microbial biomass that may be used in proteinrich food for humans and / or animals, often replacing conventional sources of protein supplementation such as soymeal or fishmeal. To produce a single cell protein, or other product, the process may comprise additional separation, processing, or treatments steps. For example, the method may comprise sterilizing the microbial biomass, centrifuging the microbial biomass, and / or drying the microbial biomass. In certain embodiments, the microbial biomass is dried using spray drying or paddle drying. The method may also comprise reducing the nucleic acid content of the microbial biomass using any method known in the art, since intake of a diet high in nucleic acid content may result in the accumulation of nucleic acid degradation products and / or gastrointestinal distress. The single cell protein may be suitable for feeding to animals, such as livestock or pets. In particular, the animal feed may be suitable for feeding to one or more animal selected from beef cattle, dairy cattle, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo / bison, llamas, alpacas, reindeer / caribou, camels, bantengs, gayals, yaks, chickens, turkeys, ducks, geese, quail, guinea fowl, squabs / pigeons, fish, shrimp, crustaceans, cats, dogs, and rodents. The composition of the animal feed may be tailored to the nutritional requirements of different animals. Furthermore, the process may comprise blending or combining the microbial biomass with one or more excipients.Culture and Fermentation

[0049] Bioreactor fermentations typically begin with preparation of the medium for growing the microorganisms, the inoculum. An inoculum refers to any small volume of a microorganism that, when added to nutrient medium, initiates the growth of millions of the same microorganism. Inoculum development refers to the preparation of a population of microorganisms from a dormant stock culture to an active state of growth that is suitable for inoculation in a larger bioreactor for production of a product.

[0050] Typically, the culture / fermentation is performed in a bioreactor. The term “bioreactor” includes a culture / fermentation device consisting of one or more vessels, towers, or piping arrangements, such as a continuous stirred tank reactor (CSTR), immobilized cell reactor (ICR), trickle bed reactor (TBR), bubble column, gas lift fermenter, static mixer, or other vessel or other device suitable for gas-liquid contact. In some embodiments, the bioreactor may comprise a first growth reactor and a second culture / fermentation reactor. The substrate may be provided to one or both reactors. Herein, the terms “culture” and “fermentation” are used interchangeably. These terms encompass both the growth phase and product biosynthesis phase of the culture / fermentation process.

[0051] The culture is generally maintained in an aqueous culture medium that contains nutrients, vitamins, and / or minerals sufficient to permit growth of the microorganism. Suitable media are known in the art.

[0052] The culture / fermentation should desirably be carried out under appropriate conditions for production of desired products. The culture / fermentation of the disclosure is performed under aerobic conditions. Reaction conditions to consider include pressure (or partial pressure), temperature, gas flow rate, liquid flow rate, media pH, dissolved gas concentration, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum gas substrate concentrations to provide that gas in the liquid phase does not become limiting, and increased product concentrations to avoid product inhibition. In particular, the rate of introduction of the substrate may be controlled to provide that the concentration of gas in the liquid phase does not become limiting.

[0053] Operating a bioreactor at elevated pressures allows for an increased rate of gas mass transfer from the gas phase to the liquid phase. Accordingly, it is generally preferable to perform the culture / fermentation at pressures higher than atmospheric pressure. Also, since a given gasconversion rate is, in part, a function of the substrate retention time and retention time dictates the required volume of a bioreactor, the use of pressurized systems can greatly reduce the volume of the bioreactor required and, consequently, the capital cost of the culture / fermentation equipment. This, in turn, means that the retention time, defined as the liquid volume in the bioreactor divided by the input gas flow rate, can be reduced when bioreactors are maintained at elevated pressure rather than atmospheric pressure. The unique reaction conditions will depend partly on the particular microorganism used. However, in general, it is preferable to operate the fermentation at a pressure higher than atmospheric pressure. Also, since a given gas conversion rate is in part a function of substrate retention time and achieving a desired retention time in turn dictates the required volume of a bioreactor, the use of pressurized systems can greatly reduce the volume of the bioreactor required, and consequently the capital cost of the fermentation equipment.

[0054] In one nonlimiting embodiment, a microorganism is produced via fermentation under conditions in which the microorganism grows and / or multiplies. For example, for an aerobic microorganism such as Cupriavidus, the microorganism may be cultured at room temperature at pH conditions of from about 6.4 to about 6.8 with mixing either by agitation / mechanical means or by a loop or air-lift reactor. As will be understood by the skilled artisan upon reading this disclosure, such exemplary fermentation conditions are in no way limiting to this invention and alternative fermentations conditions routinely determined by the skilled artisan may be used for various organisms and applications.

[0055] In one nonlimiting embodiment, the fermentation is conducted in a batch mode or fed batch mode. In another nonlimiting embodiment, the fermentation is conducted in a continuous mode.

[0056] In an embodiment, the bioreactor is a forced circulation, external loop airlift reactor. It consists of a large column known as the riser and smaller column known as the downcomer, as well as an expanded section at the top of the riser, known as the separator, where gas bubbles are separated from the liquid broth. Forced circulation is available via a pump, for instance an axial pump, at the bottom of the downcomer. Forced circulation is required to achieve suitable hydrodynamic conditions for mixing and mass transfer inside the bioreactor. The speed of the circulating pumps is manually controlled. In one embodiment, the liquid broth is pumped into the headspace through an inlet and a distribution apparatus such as a showerhead. In one embodiment, the bioreactor comprises liquid inlets for introducing nutrients, base, etc.

[0057] The feed gasses provide the carbon and energy for the microorganism in the broth. Since, O2 and H2 and to a lesser extent CO2 have limited solubility in water, these gases are introduced in the form of bubbles which provide sufficient gas-liquid mass-transfer to support the microbial growth. The primary function of the bioreactor is to create a well -controlled environment for the microbe to grow through the control of parameters such as gas flow rates, operating pressure, broth temperature, nutrient solution flow rate, pH (maintained by the addition of a base), etc. In a circulating loop bioreactor, gas is introduced along the riser section, and it rises through the column until it reaches the separator, where process tail gas is separated from the broth. From the separator, the broth flows down the downcomer section to the bioreactor circulation pump.

[0058] In an embodiment, the bioreactor contains a liquid nutrient medium (or broth) which comprising a culture of at least one microorganism. To the liquid medium or broth there are introduced various gases which the microorganism ferments to biomass, i.e. growth of the microorganism. The microorganism can also produce additional products. The gases comprise a first gaseous stream comprising O2, a second gaseous stream comprising H2, and a third gaseous stream comprising CO2. In an embodiment, the second and third gas streams are combined into a blended gas stream and provided to the bioreactor. Whether the second and third gas streams are supplied individually or combined into one stream, the second and third gaseous streams are fed to the bioreactor at a flow rate to provide the H2 and CO2 to the bioreactor at a H2: CO2 molar ratio within a predetermined molar ratio range. The range of the H2: CO2 molar ratio in the bioreactor is maintained at a value such that the microorganism uptakes the H2 and CO2 at a predetermined value. It has been found that if the H2 and CO2 gaseous streams or substrates are fed to the bioreactor at a molar ratio of H2:CC>2 of about 7.5: 1 to 4.5: 1 or about 7: 1 to 4.5: 1 or about 6.5: 1 to 4.5: 1 or about 7: 1 to 5: 1 or about 7: 1 to about 5.5: 1, then the uptake of H2 and CO2 by the microorganism will be in a H2:CC>2 molar ratio from about 6: 1 to about 4: 1 or about 6: 1 to about 4.5: 1 or about 5.5: 1 to about 4.5: 1 or about 5: 1 to about4.5: l or about 5: 1 to about 4: 1 or about 4.5:1 to about 4: 1. More specifically, the molar ratio of H2:CC>2 being fed to the bioreactor can be about 7.5: 1, or about 7: 1, or about 6.5: 1, or about 6: 1, or about 5.5:1, or about 5: 1 or about 4.5: 1. Correspondingly, the microorganism can uptake the H2 and CO2 at a molar ratio of H2:CC>2 of about 6: 1, or about 5.5: 1, or about 5: 1, or about 4.5:1 or about 4:1. Because of the high price of hydrogen, it is advantageous to operate at the lower H2:CC>2 ratios.

[0059] In an embodiment, the method comprises feeding the second gas stream comprising H2 at a flow rate or feed rate where the hydrogen is in excess of or greater than the consumption rate of H2 by the microorgansim to dilute the O2 concentration in the headspace above the fermentation broth. The feed rate or flow rate of the hydrogen in the second gas stream may be from about 5% to about 10% higher than the consumption rate of H2. Consumption rate of H2 may be calculated by several methods, such as the feed rate of H2 minus the tail gas flow rate of H2, after subtracting any diluent added to the headspace. Tail gas flow rate of H2 may be calculated by several methods, including the tail gas flow rate times the concentration of H2 in the tail gas. The feed rate of H2 may be calculated by several methods, including but not limited to the flow rate of the second gas stream comprising H2 (or by correlation with electricity consumption for green H2) plus the flow rate of the second gas stream multiplied by the concentration of hydrogen in diluted / mixed streams. Introducing excess H2 into the bioreactor results in O2 being the limiting reactant and thus smaller amounts of O2 will be present in the headspace. This controlled dilution reduces the risk of high O2 levels in the headspace especially localized high O2 concentrations. In turn this reduces the probability of explosions and allows for more latitude in safety controls. Additionally, maintaining a balanced oxygen environment, provides for the microorganisms to efficiently metabolize substrates into the desired products, thereby improving overall process yields and reliability.

[0060] In this embodiment where excess H2 is fed to the bioreactor, the outlet gas stream will comprise increased amounts of H2 versus the case where a stoichiometric amount of H2 is fed. Since H2 is expensive it is most cost effective to fully utilize hydrogen. Therefore, the outlet stream from the top of the bioreactor is compressed and recycled back to the bioreactor. At least a portion of the compressed outlet stream is recycled either by combining it with the second or third gaseous stream (or combined with the blended stream of the second and third gas streams) or sparged directly into the liquid nutrient medium. Recycling provides for the hydrogen being fully utilized in subsequent reactions within the bioreactor, maintaining a consistent and efficient hydrogen level. This sustainable approach not only enhances process efficiency but also aligns with environmental goals by reducing waste and maximizing resource utilization. Overall, integrating excess hydrogen feeding with recycling the outlet gas stream creates a robust and economical system for conducting safe and effective aerobic fermentation processes.

[0061] The oxygen is fed to the bioreactor to provide O2 at a concentration necessary for the microorganism to uptake and ferment with the H2 and CO2 to produce microbial biomass and / orat least one chemical product. The amount of oxygen fed to the bioreactor is determined by measuring the oxygen concentration in the headspace in the bioreactor. The concentration of O2 in the headspace is directly related to how much O2 the microorganism is uptaking and using to produce biomass or other products. The headspace is above the top of the liquid and is formed by passing the liquid nutrient medium through a separator as described above which disengages most of the entrained gases from the liquid portion. The headspace is thus located above the separator. Measuring the oxygen concentration can be done by using an oxygen sensor either in the headspace or in an outlet gas sample conduit or in an outlet gas conduit both of which are located approximate to the top of the reactor. Although this embodiment discloses the use of one oxygen sensor in the various locations, more than one oxygen sensor, e.g. 2, 3, or more, can be used. The use of multiple sensors has the advantage in that the readings can be averaged to provide a more accurate measurement of the oxygen concentration, or one can determine if one sensor is not reading correctly. Whether the oxygen concentration is measured in the head space or in the outlet sample gas conduit or in the outlet gas stream conduit, the value of the oxygen concentration is transmitted to a control system. Transmission of the value of the oxygen concentration can be done electrically by hardwiring the sensor with the controller or other means such as Bluetooth, Wi-Fi, etc. The control system compares the measured oxygen concentration to a predetermined value and if the oxygen concentration is above or below the predetermined value reducing or increasing the flow rate of the first gaseous stream comprising oxygen. The flow rate of the first gaseous stream is respectively reduced to operate under safety limits or is increased as per the requirement to increase production of biomass and / or other products.

[0062] In an embodiment the predetermined value of the O2 in the headspace is lower than limiting oxygen concentration (LOC) for flammability. Hydrogen is highly flammable, and even small amounts (approximately 5 vol %) can lead to explosive mixtures when combined with oxygen. According to the American National Fire Protection Association (NFPA), Limiting Oxygen Concentration (LOC) i.e., vol % O2 above which deflagration can take place, for hydrogen is approximately 5%. Limiting Oxygen Concentration for methane is approximately 12 vol %. As defined by NFPA, deflagration is propagation of a combustion zone at a velocity that is less than the speed of sound in the unreacted medium. In an embodiment, the predetermined value of O2 in the reactor headspace is defined based on the LOC, as per NFPA guidelines. For hydrogen thepredetermined concentration of O2 in the head space can be from about 1 vol % to about 2 vol % or from about 1.25 vol % to about 2 vol % or from about 1.5 vol % to 2 vol % of oxygen.

[0063] In an embodiment, the method further comprises introducing a diluent selected from H2, CO2, N2, Ar or any combination thereof into the bioreactor headspace. The reason for doing this is to dilute the concentration of the gases in the head space, especially the concentration of O2. This provides another safety feature in case the O2 concentration in the feed to the bioreactor increases suddenly resulting in a consequential spike in the O2 concentration in the head space.

[0064] In an embodiment the diluent stream along with the other gases in the headspace which comprise H2, CO2, and O2 is recycled back to the bioreactor. Recycling this outlet gas stream saves money because H2, which is very expensive, can be reused to provide energy to the microorganism and increases the production of biomass or other products. This is especially true when the diluent is H2 or comprises H2 plus another diluent such as CO2. In an embodiment, the outlet gas stream is compressed to generate a compressed outlet gas stream and at least a portion of the compressed outlet gas stream is recycled either by combining it with the second or third gaseous stream (or combined with the blended stream of the second and third gas streams) or sparged directly into the liquid nutrient medium.

[0065] In an embodiment, the diluent stream comprises CO2 and its flow is adjusted such that the total concentration of CO2 from the diluent and third gaseous stream or the CO2 in the blended gas stream is sufficient to provide the gaseous substrate at the H2: CO2 molar ratio within the predetermined molar ratio range.

[0066] In an embodiment, the diluent stream is sufficient to provide the gaseous substrate comprising CO2 at the H2: CO2 molar ratio within the predetermined molar ratio range such that no third gaseous stream is required. In such an embodiment, the outlet gas stream is compressed and recycled into the liquid nutrient medium, e.g. using the conduit which would deliver the third gaseous stream, and no third gaseous stream is required resulting in an efficient and cost-effective method for aerobic fermentation.

[0067] In an embodiment, the diluent stream comprises H2 and its flow is adjusted such that the total concentration of H2 from the diluent and second gaseous stream or the H2 in the blended gas stream is sufficient to provide the gaseous substrate at the H2: CO2 molar ratio within the predetermined molar ratio range or provide H2 in excess of the predetermined the H2: CO2 molar ratio.

[0068] In an embodiment, the diluent stream comprising H2 is sufficient to provide the gaseous substrate comprising H2 at the H2: CO2 molar ratio within the predetermined molar ratio range such that no second gaseous stream is required. As stated above, the diluent stream is also capable of providing H2 in excess of the predetermined the H2: CO2 molar ratio such that no second gaseous stream is required. In such an embodiment, the outlet gas stream is compressed and recycled into the liquid nutrient medium, e.g. using the conduit which would deliver the second gaseous stream, and no second gaseous stream is required resulting in an efficient and cost-effective method for aerobic fermentation.

[0069] In an embodiment, the diluent stream comprises H2 and CO2 and its flow is adjusted such that the total concentration of H2 and CO2 from the diluent and second plus third gaseous stream or the H2 and CO2 in the blended gas stream is sufficient to provide the gaseous substrate at the H2: CO2 molar ratio within the predetermined molar ratio range.

[0070] In an embodiment, the diluent stream comprising H2 and CO2 is sufficient to provide the gaseous substrate comprising H2 and CO2 at the H2: CO2 molar ratio within the predetermined molar ratio range such that no second and / or gaseous stream is required. In such an embodiment, the outlet gas stream is compressed and recycled into the liquid nutrient medium, e.g. using the conduit which would deliver the second and / or gaseous stream, and no second and / or third gaseous stream is required resulting in an efficient and cost-effective method for aerobic fermentation

[0071] In an embodiment, the method further comprises passing the outlet gas stream either before or after compressing it to a separation unit to separate H2 and / or CO2 from the outlet gas stream and produce a concentrated stream comprising H2 and / or CO2. The separation process also produces a N2 and O2 containing stream which is vented. The separation unit is selected from a Pressure Swing Adsorption (PSA) unit or a selective membrane unit. Selective membrane units are known which can separate both H2 and / or CO2 from the outlet gas stream. Typically, these membranes are organic membranes fabricated from polymers. Examples of these include but are not limited to poly vinylidene fluoride (PVDF), polyamide, polyimide, cellulose acetate, polystyrene, polysulfone, and polyethersulfone, polyacrylonitrile. Inorganic membranes such as ceramic or zeolitic membranes and metallic membranes, e.g. palladium, can also be used. By using mixed matrix membranes both H2 and CO2 can be separated from the outlet gas stream.

[0072] PSA systems can be used to separate hydrogen or CO2 from the outlet gas stream. Since hydrogen is expensive a PSA system which separates the hydrogen from the outlet gas stream isused. In an embodiment, the outlet stream from the hydrogen separation PSA system comprising CO2, O2 and N2 can be passed to a second PSA system where the CO2 is separated from the other gases. If only the hydrogen PSA system is used, then the concentrated hydrogen stream is recycled to the bioreactor (with or without compression). The concentrated hydrogen stream can be fed as part of the second gas stream or into the blended stream or directly into the fermentation broth using an additional inlet. When both hydrogen and CO2 streams are produced, they can be blended before recycling to the bioreactor. Usually, the hydrogen stream is blended with a portion of the CO2 stream to provide the CO2 to the bioreactor to achieve the desired H2: CO2 predetermined ratio. Producing a concentrated H2 and CO2 stream and recycling it to the bioreactor smaller amounts of H2 and CO2 need to be fed using the second, third or blended gas stream resulting in an economic benefit. Removing oxygen from the recycled stream also improves the overall safety of the process. Finally, removing inert gases such as nitrogen improves the efficiency of the system since inert compounds would not be increasing either in the fermentation liquid or the headspace. If concentration of H2 and CO2 from the outlet stream is not carried out, then only a portion of the outlet stream can be recycled to minimize accumulation of inert components in the bioreactor. This again results in an economic advantage since expensive hydrogen gas is not wasted.

[0073] In an embodiment, wherein multiple bioreactors are used they all share a single compressor. In another embodiment multiple bioreactors have respective compressors, one each for an individual bioreactor. By way of an example, multiple bioreactors are used when there is one or more bioreactor for growth of microorganism and one or more bioreactor for product production. When multiple bioreactors are used, the compressed outlet gas is recycled either to any one of the bioreactors or to all the bioreactors. The step of compressing the outlet gas stream to generate a compressed outlet gas stream improves overall efficiency of the fermentation process and makes it cost effective.

[0074] In an embodiment, the compressor for the outlet gas stream is a liquid ring compressor. Conventionally, a liquid ring compressor is used for vacuum applications while in the present application the liquid ring compressor is used in a positive pressure application. The liquid ring compressor reduces the risk of internal spark formation within the compressor as compared to normal gas compressor with very tight tolerances, rotating at very high rotations per minute. Utilisation of the liquid ring compressor has an advantage of providing an additional safety feature to the present apparatus. Another advantage associated with liquid ring compressor is that it isrobust and can handle droplets of liquid or foam contained in the outlet gas stream. The outlet gas coming out of the bioreactor is a foamy liquid and the majority of compressors cannot handle droplets of liquid or foam getting into them. Since the liquid ring compressor has a recirculating liquid loop, presence of liquid droplets or foam in the outlet gas will not be a problem.

[0075] In an embodiment, the second gaseous substrate comprising H2 is supplied from a H2 source selected from a water electrolysis process, a hydrocarbon reforming process, a hydrogen purification process, a solid biomass gasification process, a solid waste gasification process, a coal gasification process, a hydrocarbon gasification process, a methane reforming process, a refinery tail gas, a plasma reforming process, a partial oxidation process, or any combination thereof.

[0076] In one embodiment, hydrogen is produced in a water electrolyser. Water is introduced to the electrolyser which may receive power, for example 4.78kwh / Nm3, from a power source, to convert water into hydrogen and oxygen according to the following stoichiometric reaction: H2O + electricity -> 2 H2 + O2 + heat

[0077] Water electrolysis technologies are known, and exemplary processes include alkaline water electrolysis, proton exchange membrane (PEM) electrolysis, and solid oxide electrolysis. Suitable electrolysers include alkaline electrolysers, PEM electrolysers, and solid oxide electrolysers. The oxygen generated as a by-product of water electrolysis may be employed for various purposes including feeding it to the bioreactor.

[0078] In an embodiment, a diluent gas may be employed in the headspace to dilute and or flush the headspace gasses when needed, especially to keep within a set limit. The diluent gas may function as a purge gas. The diluent gas may be continuous or employed only when needed, or the flowrate may be adjusted depending upon the need to control oxygen content in the headspace. In an embodiment, the gasses in the headspace are well mixed including via mixing or back mixing, such as with a diluent gas, so that the volume of the oxygen gas in the head space is always lower than a predetermined safe limit. If the diluent gas is not well mixed with the headspace gasses, the flowrate of the diluent gas would be much higher than if the gasses were well mixed. The diluent gas is to mix with the headspaces gases across the diameter of the reactor, and the larger the diameter of the reactor, the greater diameter the diluent gas would need to well mix across. The response time of the diluent gas system to maintain the level of oxygen in the head space below a preset level is a valuable feature. The faster the response time, the greater the likelihood of any oxygen remaining below the preset limit even in the event of a process upset.

[0079] An added benefit is to improve the mixing of the gasses in the head space and thereby the response time of a purge, by using the recycle fluid in a strategic shower pattern or configuration. The recycle fluid may contain recycled broth and optionally, antifoaming agent(s). The mixing may or may not involve a diluent gas being used as a purge.

[0080] Liquid broth may be recycled through an external liquid recycle loop for example by pumping from the bottom section of the bioreactor to the top section of the bioreactor, such as to the headspace of the bioreactor. The recycle liquid is introduced into the headspace of the bioreactor by means of liquid distribution devices, such as shower heads or spray nozzles. The liquid streams or droplets formed by these distribution devices then contact the gas, in the form of bubbles, that becomes disengaged at gas / liquid interfaces and continues flowing upwardly (in bulk) through the headspace. The spray action contributes to gentle mixing of the headspace gases, ensuring uniform distribution and preventing the formation of high concentration gas pockets. It also contributes to gentle mixing of the liquid with any foam that may have formed thus breaking up the foam. This prevents the foam from accumulating and becoming problematic. In this manner, the bioreactor operates with counter current gas and liquid flows, upwardly flowing gas and downwardly flowing liquid, in the headspace, which is disposed above the liquid phase. The diluent gas may be input into the reactor at a location above the fluid level, but below the introduction of the recycle fluid where the recycle fluid is introduced in one, two, three, four, or more shower heads. In an embodiment, 6 to 10 shower heads would be employed. Multiple recycle shower heads may be arranged in a grid pattern, such as 4 or more shower heads arranged in a circular, square, or rectangular pattern near to the center of the reactor, but spaced apart so as to increase the surface area of the aggregate of the mixing power of the showers. Recycle fluid exiting the shower heads are in the form of droplets that fall through the headspace gas and diluent gas mixture. The momentum of the droplets is transferred to the gas and drives circulation. For example, when the velocity magnitude has a peak of about 8 m / sec for the liquid drops the resulting motion of the gas is substantially increased. The enhanced mixing zone correspond to the height of the shower and the distance from the shower head to the fluid level. Typically, showers are used to control foam by using the momentum of the droplets from the shower head to break bubbles that make up foam. In an embodiment, by placing the shower heads strategically in the headspace of the reactor, both the benefits of foam control and enhanced mixing may be achieved.

[0081] In an embodiment the shower heads are positioned at the top of the reactor and therefore the top of the headspace, and the strategic positioning refers to how multiple shower heads are arranged cross sectionally over the diameter of the reactor. It is the pattern of the multiple shower heads at the top of the reactor along with the velocity of the droplets that results in the enhanced mixing of gases in the headspace. Traditionally, one or more shower heads are positioned to provide a uniform distribution of shower droplets across the diameter of the reactor. Surprisingly, it is a nonuniform distribution that is needed for the enhanced mixing. A balance is therefore struck so that a near-uniform distribution is achieved at the liquid interface between the reactor fluid and the headspace which addresses foam control, but heterogeneity is created in the headspace above the liquid interface to drive mixing in the gas phase. A location pattern of shower heads for foam control only might look like evenly spaced shower heads across the cross section of the reactor, while a pattern of shower heads that would be effective for both foam control and enhanced mixing might have the shower head locations concentrated more to the center of the reactor and not near to the periphery of the reactor. In an embodiment, the shower heads may be smaller than traditionally employed but not pointing straight down (parallel to the vertical axis), but each slightly offset from the vertical axis of the reactor. About ten to fifteen degrees offset from the vertical access of the reactor is suitable. The offset may be angled to direct the recycle fluid toward the sidewalls of the reactor as opposed to towards the vertical axis center of the reactor, or vice versa. In an embodiment as shown in Fig. 4, a ring of 6 to 10 shower heads, 410, are positioned at the top of reactor 400 and so that each shower head in the ring is about half the distance from the center axis of the reactor to the sidewall of the reactor. Each shower head is offset from the vertical axis of the reactor to direct recycle fluid to the sidewalls of the reactor as described above. An exemplary offset is shown as 420. In an embodiment where the ring of shower heads has a diameter so that each shower head is located about two-thirds the distance from the center axis of the reactor to the sidewall, the shower heads are angled from about 10 to about 15 degrees from the vertical axis to direct recycle fluid in the direction toward the center axis of the reactor. Another shower head may be located at the top and in the center of the cross section of the reactor, 410. Spray nozzles are a suitable form of shower heads.

[0082] In an embodiment, where at least one spray nozzle is used, the method further comprises measuring a differential pressure across the spray nozzle, inputting the measured differential pressure to a differential pressure control system and comparing the measured value with apredetermined setpoint value and increasing or decreasing the liquid nutrient medium flow rate if the measured differential pressure is below or above the predetermined setpoint value. By measuring the differential pressure across the spray nozzle, the flow rate of the liquid being sprayed is determined. This provides for a sufficient amount of liquid being delivered to the bioreactor headspace to break up bubbles and / or foam. Measuring differential pressure is an inexpensive way of monitoring flow rate as compared to utilizing a flow meter, although flowmeters can also be used for this control system. Maintaining a consistent differential pressure provides for the spray pattern and distribution of gases in the headspace remaining uniform. This aids in maintaining suitable conditions within the bioreactor headspace by not creating pockets of high concentration of gases which may form a highly explosive mixture. If pockets of high concentrations of O2 (above the predetermined value) and H2 are created, they may not be reflected in the concentration of O2 measured by the oxygen sensors in the headspace or outlet gas sample conduit or outlet gas stream conduit. In this case, an explosive mixture can be created before an operator has an opportunity to adjust the flow of O2. By providing efficient mixing, the O2 sensors measure the true concentration of O2 throughout the head space thus providing additional time for an operator to adjust the O2 flow rate. If the differential pressure across the spray nozzle shows low or no flow of liquid nutrient medium, the flow of the first, second and third gaseous streams to the bioreactor is reduced or shut off respectively by the control system.

[0083] In an embodiment, instead of feeding H2 and CO2 as the gaseous substrate, CH4 is used as the gaseous substrate. In this case, the microorganism ferments the CH4 and O2 to produce microbial biomass and products such as lipids. As also described above, a headspace which comprises O2 and CH4 is formed by passing the liquid nutrient medium through a separator as described above which disengages most of the entrained gases from the liquid portion. The headspace is thus located above the liquid nutrient medium and the separator. In an embodiment, the method further comprises introducing a diluent comprising CO2 in the bioreactor headspace. The CH4 is fed at a flow rate to maintain the O2 concentration in the headspace below a limiting oxygen concentration (LOC) for methane. LOC is the concentration of oxidant in a fuel-oxidant- diluent mixture below which a deflagration cannot occur under specified conditions. As per NFPA 69 (2024) the technique for oxidant concentration reduction for deflagration prevention is permitted to be considered where a mixture of oxidant and flammable mixture is confined within an enclosure.

[0084] In an embodiment the predetermined value of the O2 in the headspace is kept below than limiting oxygen concentration (LOC) for flammability. For example, the updated LOC for methane, as per Table C.l (a) in NFPA 69 (2024) is 13.1vol% in CCh-Air mixture. Additional safety margin of 2 volume percent below the worst credible case LOC, as prescribed by NFPA is applied to obtain the operating LOC. It is desirable to have the oxygen concentration in the headspace, i.e. predetermined value, to be in a range from about 5 vol. % to about 11 vol. % or from 6 vol. % to about 11. vol % or 5 vol. % to 10 vol. % or from about 6 vol. % to about 10 vol. %. To meet the continuous monitoring and control objectives, the gaseous oxygen concentration in the outlet gas stream, or the outlet gas sample stream or the bioreactor headspace is measured and transmitted to a control system which compares the measured oxygen concentration to a predetermined value and if the oxygen concentration is above the predetermined value reduces the flow of the O2 gaseous stream to the bioreactor.

[0085] The method further comprises recycling a portion of the formed liquid broth from the bottom of the bioreactor to the top of the bioreactor headspace wherein the portion of the liquid broth is introduced into the bioreactor headspace by at least one spray nozzle. The differential pressure across the nozzle is measured, and input to a differential pressure control system which compares the measured value with a predetermined setpoint value and increasing or decreasing the liquid nutrient medium flow rate if the measured differential pressure is below or above the predetermined setpoint value. If the differential pressure across the spray nozzle shows low or no flow of liquid nutrient medium, the flow of the first and second gaseous streams to the bioreactor is stut off.

[0086] The bioreactor is configured to provide enough mass transfer to allow the microorganism(s) to access the H2, CO2, CH4 and O2. In an embodiment, the apparatus comprises a bioreactor vessel comprising an exterior casing having a top and a bottom, the bioreactor vessel containing a liquid nutrient medium comprising a culture, a headspace overlying the liquid; the bioreactor vessel having at least one gas comprising O2 inlet located on the exterior casing and below the top surface of the liquid nutrient medium and in fluid communication with a gas comprising O2 supply conduit in fluid communication with a gas comprising O2 supply unit, the gas comprising O2 supply conduit further comprising a gas comprising O2 flow control valve; the bioreactor vessel having at least one gaseous substrate inlet located on the exterior casing and below the top surface of the liquid nutrient medium and in fluid communication with at least onegaseous substrate supply conduit, the gaseous substrate supply conduit in fluid communication with a gaseous substrate supply unit, the conduit further comprising a gaseous substrate flow control valve; an outlet gas outlet located approximate to the top of the bioreactor vessel and within the headspace, and in fluid communication with at least one outlet gas sample conduit comprising at least one oxygen sensor and optionally at least one oxygen sensor located within the headspace, the at least one oxygen sensors in the headspace or on the outlet gas sample conduit or on the outlet gas conduit to measure a concentration of oxygen in the headspace and are in communication with a control system; and the control system controls the flow of O2 from the gas comprising O2 supply unit to the bioreactor; wherein the outlet gas conduit is in fluid communication with a liquid ring compressor in fluid communication with compressed outlet gas conduit, wherein the compressed outlet gas conduit is in fluid communication with the gaseous substrate supply conduit or the bioreactor; and the headspace comprising a spray nozzle to introduce a portion of the liquid nutrient medium from the bioreactor into the headspace.

[0087] In an embodiment, the bioreactor vessel comprises at least one diluent inlet located above the top surface of the liquid nutrient medium in the gas headspace and in fluid communication with at least one diluent conduit, the diluent conduit in fluid communication with a diluent supply unit. The diluent supply unit supplies a diluent selected from H2, CO2 N2, Ar, , or any combination thereof. The diluent is continuously fed into the headspace of the bioreactor vessel to dilute the concentration of O2 in the head space. In a preferred embodiment, the diluent supply unit supplies a gas comprising CO2. In an embodiment, the diluent stream is sufficient to provide the gaseous substrate comprising CO2 at the H2: CO2 molar ratio within the predetermined molar ratio range such that no third gaseous stream is required. In such an embodiment, the outlet gas stream is compressed and recycled into the liquid nutrient medium, e.g. using the conduit which would deliver the third gaseous stream and no third gaseous stream is required resulting in an efficient and cost-effective method for aerobic fermentation.

[0088] In specific embodiments, as illustrated in the Figure a first gaseous substrate supply unit supplies a gas comprising O2 as the first gaseous substrate; the second supply unit supplies a second substrate which is a gas comprising H2 and the third supply unit supplies a third substrate which is a gas comprising CO2.

[0089] In another embodiment, when one of the gaseous substrates is CH4 only two gaseous substrate supply units are provided such that a first unit supplies a gas comprising O2 as the firstgaseous substrate; and the second supply unit supplies a second substrate which is a gas comprising CH4.

[0090] The Figure illustrates a bioreactor vessel (100) comprising an external casing having a top (600) and bottom (700) for aerobic gas fermentation in accordance with an embodiment. Individual gases, a gas comprising O2, and either a gas comprising H2 as well as a gas comprising CO2, or a gas comprising CH4enter the bioreactor at different and separate inlets located on the external casing of the bioreactor vessel and below the level of the fermentation broth or medium.

[0091] A gas comprising O2 is provided by gas supply unit (10) in fluid communication with a gas comprising O supply conduit (18) which delivers the gas comprising O2 to bioreactor vessel (100) through an inlet on the exterior casing of the bioreactor vessel. The gas comprising O2 can enter at one inlet (separate from the other gases) or at multiple inlet points on the vessel.

[0092] The supply conduit (18) comprises at least one oxygen sensor to measure the concentration of oxygen in the stream flowing through the conduit, and one flow meter (not shown) to measure the flow of oxygen into the system. The gas comprising O2 supply conduit (18) further comprises at least one gas comprising O2 control valve (114). The oxygen sensor, O2 flow meter and the O2 control valve (114) are in communication with the control system so as to increase or decrease the flow rate of the first gaseous stream comprising O2 based on the comparison of the measured oxygen concentration in the headspace to a predetermined setpoint value. This is to prevent a sudden increase in O2 concentration in the bioreactor system if the concentration of O2 in the feed gas suddenly rises.

[0093] As shown in the Figure, in one embodiment where the gaseous substrate comprises a gas comprising H2 and a gas comprising CO2, the gaseous substrate supply unit is split into two different units (20, 30). One unit (20) supplies H2 gas to the bioreactor while the other (30) supplies a gas comprising CO2. A gas comprising H2 supply unit (20) is in fluid communication with a gas comprising H2 supply conduit (32) which delivers the gas comprising H2 to bioreactor vessel (100) through an inlet on the external casing of the bioreactor vessel. The supply conduit (32) comprises at least one H2 sensor to measure the concentration of hydrogen flowing through the conduit, and one flow meter (not shown) to measure the flow of hydrogen into the system. The gas comprising H2 supply conduit (32) further comprises at least one gas comprising H2 control valve (122). The H2 sensor, H2 flow meter and the H2 control valve (122) are in communication with the controlsystem. This is to prevent a sudden increase or decrease in H2 concentration in the bioreactor system if the concentration of H2 in the feed gas suddenly rises or falls.

[0094] As shown in the Figure, when the other gaseous substrate is a gas comprising CO2, gaseous substrate supply unit (30) is a gas comprising CO2 supply unit which is in fluid communication with a gas comprising CO2 supply conduit (32) which delivers the gas comprising CO2 to bioreactor vessel (100) through an inlet on the external casing of the bioreactor vessel. The supply conduit (22) comprises at least one CO2 sensor which measures the concentration of the CO2 flowing through the conduit, and one flow meter (not shown) to measure the flow of CO2 into the system. The gas comprising CO2 supply conduit (22) further comprises at least one gas comprising CO2 control valve (112). The CO2 sensor, CO2 flow meter and the CO2 control valve (112) are in communication with the control system. This is to prevent a sudden increase or decrease in CO2 concentration in the bioreactor system if the concentration of CO2 in the feed gas suddenly rises or falls.

[0095] In an alternate embodiment, the gas comprising H2 and gas comprising CO2 can be supplied to the bioreactor using one supply conduit (22 or 32), one gas inlet to the vessel (and one sparger), one flow control valve (112 or 122), one flow controller (not shown), one flow transmitter (not shown). However, sensors for H2 and CO2 would be provided on the chosen conduit. Either one or two supply units (20 or 30) can be used to provide the two gases.

[0096] When the gaseous substrate is methane, only one CH4 supply unit (20 or 30), one CH4 supply conduit (22 or 32), one CH4 inlet to the vessel, one CH4 flow control valve (112 or 122), one CH4 flow meter and one CH4 sensor is provided.

[0097] In another embodiment the bioreactor further comprises a diluent supply unit (500) in fluid communication with a diluent supply conduit which delivers the diluent to the bioreactor vessel (100) through an inlet located above the top surface of the liquid nutrient medium in the gas headspace. The diluent supply unit supplies a gas comprising CO2.

[0098] The bioreactor (100) contains a liquid culture medium and a gas headspace above the top of the liquid culture medium to the top of the vessel. As also described above, a headspace which comprises O2 is formed by passing the liquid nutrient medium through a separator as described above which disengages most of the entrained gases from the liquid portion. The headspace is thus located above the liquid nutrient medium and the separator. The bioreactor vessel (100) comprises a vent gas or outlet gas flow conduit (300), as well as at least one outlet gas sampling conduit (800)having at least one oxygen sensor (72). The outlet gas sample is typically a small slipstream of the outlet gas flow, which is sent to a safe vent (900), however this sensor can also be mounted directly on the outlet gas flow conduit (300). The outlet gas sampling conduit also has gas flow meters / transmitters for controlling and monitoring the gas flow and ensuring adequate gas flow for a high quality measurement and a fresh sample from the headspace. Although the Figure shows the use of single oxygen sensor, it is to be understood that multiple sensors can be used. The flow meters / transmitters are in communication with the control system. The oxygen sensor (72) on the outlet gas sample conduit measures the concentration of oxygen in the conduit which is equivalent to the oxygen concentration in the gas headspace. This sensor is in communication with the control system which increases or decreases the flow rate of the first gaseous stream.

[0099] The outlet gas flow conduit (300) is in fluid communication with a compressor (1000) for compressing the outlet gas. The compressed outlet gas conduit (400) is recycled (not shown) to combine with the second or third gaseous stream or directly into the liquid nutrient medium in the bioreactor

[0100] In an embodiment, at least one O2 sensor is placed in the headspace of the bioreactor vessel to measure the concentration of oxygen gas in the gas headspace. O2 sensors placed in the headspace have the same function as that of the sensors placed on the outlet gas sample conduit or the outlet gas flow conduit, however sensors placed on the outlet gas sample conduit and outlet gas flow conduit are more protected against water ingress and thus more reliable.

[0101] In an embodiment, when the gaseous substrate is a gas comprising CH4 at the outlet gas sample conduit comprises at least one CH4 sensor and optionally at least one CH4 sensor located within the gas headspace. The CH4 sensor in the gas headspace or on the outlet gas sample conduit measure concentration of CH4 in the gas headspace and are in communication with the control system.

[0102] The control system can be located anywhere, the sensors are located on the conduits entering and exiting the bioreactor where they are measuring concentration or flow, and transmitters send the concentration or flow signals via cables or other means to the control system (or directly to the controller if the controller is its own stand-alone unit). The control system used in the bioreactor system comprises several controllers and a computer processor which executes a computer program implemented process for controlling the flow rate or shutting off the gas comprising O2 based on the measured concentration of O2 in the gas headspace. The control systemor individual controllers control the flow of diluent gas from the diluent gas supply unit to the bioreactor. In an embodiment, the control system is a cascade control system wherein the headspace oxygen controller measures the headspace O2 through O2 sensors, and the manipulated variable output from that controller is the “setpoint” for the oxygen flow controller. The headspace oxygen controller gives a setpoint value to the oxygen flow controller. The oxygen flow controller then measures the flow from the oxygen sensor / transmitter and manipulates the oxygen flow control valve to achieve the setpoint it received from the headspace oxygen controller. The controllers in general measure the error between the present value (PV) and the setpoint (SP). Such difference (error = PV-SP) is constantly used to adjust the output by what is known as a proportional integral derivative (PID) controller. PID controllers are known in the art and can be configured to respond at different speeds and also to respond to trends in the error value. For example, the “integral” portion of the controller looks at the accumulated error over time, rather than the instantaneous error at the exact moment, and will cause the response to speed up or slow down as required, if the error is getting larger or smaller, and prevent overshooting the SP value. In an embodiment, the sensor, transmitter, controller, and manipulated output can be integrated in a single device, e.g., Mass Flow Controller (MFC) having internal logic systems for the flow control. Generally, MFCs are swapped out for independent meter and valve arrangements with the calculations being done by the main control system in larger applications.

[0103] In some embodiments, when a chemical is co-produced with protein, target products may be separated or purified from a fermentation broth using any method or combination of methods known in the art, including, for example, fractional distillation, evaporation, pervaporation, gas stripping, phase separation, and extractive fermentation, including for example, liquid-liquid extraction. In certain embodiments, target products are recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating microbial cells from the broth (conveniently by filtration), and recovering one or more target products from the broth. Alcohols and / or acetone may be recovered, for example, by distillation. Acids may be recovered, for example, by adsorption on activated charcoal. The cell-free permeate remaining after target products have been removed is preferably returned to the bioreactor. Additional nutrients (such as B vitamins) may be added to the cell-free permeate to replenish the medium before it is returned to the bioreactor. Purification techniques may include affinity tag purification (e.g. His, Twin- Strep, and FLAG), bead-based systems, a tip-based approach, and FPLC system for larger scale,automated purifications. Purification methods that do not rely on affinity tags (e g. salting out, ion exchange, and size exclusion) are also disclosed.

[0104] In some embodiments, the produced chemical product may be isolated and enriched, including purified, using any suitable separation and / or purification technique known in the art. In an embodiment, the produced chemical product is gaseous. In one embodiment, the chemical product is a liquid. In an embodiment, a gaseous chemical product may pass a filter, a gas separation membrane, a gas purifier, or any combination thereof. In one embodiment, the chemical product is separated by an absorbent column. In another embodiment, the chemical product is stored in one or more cylinders after separation. In one embodiment, the chemical product is integrated into an infrastructure or process of an oil, gas, refinery, petrochemical operation, or any combination thereof. The infrastructure or process may be existing or new. In an embodiment, the gas fermentation product is integrated into oil and gas production, transportation and refining, and / or chemical complexes. In another embodiment, the source of the feedstock is from an oil, gas, refinery, petrochemical operation, or any combination thereof. In an embodiment, the gas fermentation product is integrated into an infrastructure or process of an oil, gas, refinery, petrochemical operation, or any combination thereof, and the source of the feedstock is from an oil, gas, refinery, petrochemical operation, or any combination thereof

[0105] In some embodiments, distillation may be employed to purify a product gas. In an embodiment, gas-liquid extraction may be employed. In an embodiment, a liquid product isolation may also be enriched via extraction using an organic phase. In another embodiment, purification may involve other standard techniques selected from ultrafiltration, one or more

[0106] In some embodiments of the methods disclosed herein, the microorganism produces a commodity chemical product, microbial biomass, single cell protein (SCP), one or more intermediates, or any combination thereof.

[0107] In some embodiments, the microbial biomass has a unit value. In one embodiment, the microbial biomass has a market value.

[0108] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The reference to any prior art in this specification is not, and should not be taken as, anacknowledgement that that prior art forms part of the common general knowledge in the field of endeavour in any country.

[0109] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. The term “consisting essentially of’ limits the scope of a composition, process, or method to the specified materials or steps, or to those that do not materially affect the basic and novel characteristics of the composition, process, or method. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the term “about” means ±20% of the indicated range, value, or structure, unless otherwise indicated.

[0110] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, any concentration range, percentage range, ratio range, integer range, size range, or thickness range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0111] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.EXAMPLE

[0112] Cupriavidus necator microorganism in a liquid nutrient solution was placed in a closed loop reactor. To the fermentation broth there was fed air FL, and CO2 thereby producing biomass. The production rate of the biomass was monitored by measurement of optical density and of the outlet flow from the continuous bioreactor system. The system was operated at a dilution rate of 0.125 / hr. Optical density was calibrated to a dry weight measurement for this organism, theproduction rate of dry kg per day is reported. The gas feed flow rates were measured by thermal mass flow meters. Fig. 2 illustrates the biomass production over time, with time on the X-axis, left y-axis indicating feed gas flows (NLPM), and right y-axis demonstrating biomass production (kg / day). The plots shows that the H2 to CO2 feed ratio was maintained at a constant level of approximately 7:1 from days 8 to 13, and approximately 6.2: 1 from days 13 to 16. While the H2 and CO2 flows where kept constant for time intervals, the O2 flow rate was adjusted to meet the requirements of the microorganism. Such dynamic control provided for the O2 flow rate to be maintained at the maximum amount of O2 which to maximize the production rate of biomass while maintaining safe operating conditions. Fig. 3 depicts the headspace O2 smooth controller response necessary for maintaining the desired setpoint (SP). Fig. 3 further shows how the controller responded when the O2 concentration briefly went over the O2 SP. The plots in Fig. 3 present the headspace O2 controller's response to changes in O2 concentration over time. Time is plotted on the X-axis, the left Y-axis represents feed gas flowrates in NLPM, and the right Y-axis represents the headspace O2 concentration and headspace O2 concentration setpoint for the controller. The plots show that when the headspace oxygen concentration rises above the setpoint at approximately day 10.7, the controller responds swiftly with a reduction in the feed 02 flow rate to maintain a safe headspace O2 concentration. Similarly, from day 10 to day 12, as H2 and CO2 flow rates were adjusted, the microorganism was able to consume more O2, and the controller increased the O2 flow rate to maintain the O2 concentration setpoint. This allowed increased production of biomass, while maintaining safe operating conditions.Definitions

[0113] Unless otherwise defined, the following terms as used throughout this specification are defined as follows:

[0114] A “microorganism” is a microscopic organism, especially a bacterium, archaeon, virus, or fungus. In an embodiment, the microorganism of the disclosure is a bacterium.

[0115] The term “non-naturally occurring” when used in reference to a microorganism is intended to mean that the microorganism has at least one genetic modification not found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Non-naturally occurring microorganisms are typically developed in a laboratory or research facility.

[0116] The terms “genetic modification,” “genetic alteration,” or “genetic engineering” broadly refer to manipulation of the genome or nucleic acids of a microorganism by the hand of man. Likewise, the terms “genetically modified,” “genetically altered,” or “genetically engineered” refers to a microorganism containing such a genetic modification, genetic alteration, or genetic engineering. These terms may be used to differentiate a lab-generated microorganism from a naturally occurring microorganism. Methods of genetic modification of include, for example, heterologous gene expression, gene or promoter insertion or deletion, nucleic acid mutation, altered gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis methods, gene shuffling, and codon optimization. The microorganisms of the disclosure can be genetically engineered.

[0117] “Recombinant” indicates that a nucleic acid, protein, or microorganism is the product of genetic modification, engineering, or recombination. Generally, the term “recombinant” refers to a nucleic acid, protein, or microorganism that contains or is encoded by genetic material derived from multiple sources, such as two or more different strains or species of microorganisms. The microorganisms of the disclosure can be recombinant.

[0118] “Heterologous” refers to a nucleic acid or protein that is not present in the wild-type or parental microorganism from which the microorganism of the disclosure is derived. For example, a heterologous gene or enzyme may be derived from a different strain or species and introduced to or expressed in the microorganism of the disclosure. The heterologous gene or enzyme may be introduced to or expressed in the microorganism of the disclosure in the form in which it occurs in the different strain or species. Alternatively, the heterologous gene or enzyme may be modified in some way, e.g., by codon-optimizing it for expression in the microorganism of the disclosure or by engineering it to alter function, such as to reverse the direction of enzyme activity or to alter substrate specificity.

[0119] In particular, a heterologous nucleic acid or protein expressed in the microorganism described herein may be derived from Bacillus, Clostridium, Cupriavidus, Escherichia, Gluconobacter, Hyphomicrobium, Lysinibacillus, Paenibacillus, Pseudomonas, Sedimenticola, Sporosarcina, Streptomyces, Thermithiobacillus, Thermotoga, Zea, Klebsiella, Mycobacterium, Salmonella, Mycobacteroides, Staphylococcus, Burkholderia, Listeria, Acinetobacter, Shigella, Neisseria, Bordetella, Streptococcus, Enterobacter, Vibrio, Legionella, Xanthomonas, Serratia, Cronobacter, Cupriavidus, Helicobacter, Yersinia, Cutibacterium, Francisella, Pectobacterium,Arcobacter, Lactobacillus, Shewanella, Erwinia, Sulfurospirillum, Peptococcaceae, Thermococcus, Saccharomyces, Pyrococcus, Glycine, Homo, Ralstonia, Brevibacterium, Methylobacterium, Geobacillus, bos, gallus, Anaerococcus, Xenopus, Amblyrhynchus, rattus, mus, sus, Rhodococcus, Rhizobium, Megasphaera, Mesorhizobium, Peptococcus, Agrobacterium, Campylobacter, Acetobacterium, Alkalibaculum, Blautia, Butyribacterium, Eubacterium, Moorella, Oxobacter, Sporomusa, Thermoanaerobacter, Schizosaccharomyces, Paenibacillus, Fictibacillus, Lysinibacillus, Ornithinibacillus, Halobacillus, Kurthia, Lentibacillus, Anoxybacillus, Solibacillus, Virgibacillus, Alicyclobacillus, Sporosarcina, Salimicrobium, Sporosarcina, Pianococcus, Corynebacterium, Thermaerobacter, Sulfobacillus, or Sy mbi ob acterium .

[0120] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labelling component. As used herein, the term “amino acid” includes natural and / or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

[0121] ‘ ‘Mutated” refers to a nucleic acid or protein that has been modified in the microorganism of the disclosure compared to the wild-type or parental microorganism from which the microorganism of the disclosure is derived. In one embodiment, the mutation may be a deletion, insertion, or substitution in a gene encoding an enzyme. In another embodiment, the mutation may be a deletion, insertion, or substitution of one or more amino acids in an enzyme.

[0122] “Disrupted gene” refers to a gene that has been modified in some way to reduce or eliminate expression of the gene, regulatory activity of the gene, or activity of an encoded protein or enzyme. The disruption may partially inactivate, fully inactivate, or delete the gene or enzyme. The disruption may be a knockout (KO) mutation that fully eliminates the expression or activity of a gene, protein, or enzyme. The disruption may also be a knock-down that reduces, but does not entirely eliminate, the expression or activity of a gene, protein, or enzyme. The disruption may be anything that reduces, prevents, or blocks the biosynthesis of a product produced by an enzyme. The disruption may include, for example, a mutation in a gene encoding a protein or enzyme, amutation in a genetic regulatory element involved in the expression of a gene encoding an enzyme, the introduction of a nucleic acid which produces a protein that reduces or inhibits the activity of an enzyme, or the introduction of a nucleic acid (e.g., antisense RNA, RNAi, TALEN, siRNA, CRISPR, or CRISPRi) or protein which inhibits the expression of a protein or enzyme. The disruption may be introduced using any method known in the art. For the purposes of the present disclosure, disruptions are laboratory-generated, not naturally occurring.

[0123] A “parental microorganism” is a microorganism used to generate a microorganism of the disclosure. The parental microorganism may be a naturally occurring microorganism (i.e., a wildtype microorganism) or a microorganism that has been previously modified (i.e., a mutant or recombinant microorganism). The microorganism of the disclosure may be modified to express or overexpress one or more enzymes that were not expressed or overexpressed in the parental microorganism. Similarly, the microorganism of the disclosure may be modified to contain one or more genes that were not contained by the parental microorganism. The microorganism of the disclosure may also be modified to not express or to express lower amounts of one or more enzymes that were expressed in the parental microorganism.

[0124] The microorganism of the disclosure may be further classified based on functional characteristics. For example, the microorganism of the disclosure may be or may be derived from a Cl -fixing microorganism, an aerobe, an autotroph, and / or a methanotroph. The microorganism of the disclosure may be selected from chemoautotroph, hydrogenotroph, knallgas microbes, methanotroph, or any combination thereof. In some embodiments, the microorganism may be a hydrogen-oxidizing one, a knallgas one, or any combination thereof, with the capability to grow and synthesize biomass on gaseous carbon sources such as syngas and / or CO2, such that the production microorganisms synthesize targeted chemical products under gas cultivation. Knallgas microbes, hydrogenotrophs, and chemoautotrophs more broadly can capture CO2 or CO as their sole carbon source to support biological growth. In some embodiments, this growth includes the biosynthesis of amino acids and proteins. In some embodiments of the present disclosure knallgas organisms and / or hydrogenotrophs and / or other chemoautotrophic microorganisms are grown on a stream of gases including but not limited to one or more of the following: CO2; H2; along with inorganic minerals dissolved in aqueous solution. In some embodiments knallgas microbes and / or hydrogenotrophs and / or other chemoautotrophic and / or methanotrophic microorganisms convert greenhouse gases into biomolecules including amino acids and proteins. US Patent No. 11220700B2 provides a process for production of at least one product from a methane-containing feedstock. The process comprises providing a gaseous substrate comprising CH4 and O2 to a bioreactor comprising a culture of at least one methanotrophic microorganism in a liquid nutrient medium to produce at least one product such as lipids and amino acids. Said US patent is incorporated herein by reference in entirety. The lipid products are contained within the cell or in the cellular membrane. In these cases, the lipid products need to be extracted from the bacterial biomass. Therefore, following the microbial conversion process in the reactor, the biomass is fed to an extraction zone. The extraction zone may comprise multiple extraction units for different stages of the extraction process. As a first stage, the biomass may be provided to a cell disruption unit at a solids concentration amenable to pumping biomass (less than 40% solids and preferably between 1 and 10% solids) for pretreatment prior to lipid extraction. The pretreatment step may consist of either a chemical or physical treatment of the cell biomass, preferably either high-pressure homogenization, high temperature incubation (between about 50°C and about 200°C, preferably between about 75°C and about 90°C) or acid or alkaline pretreatment (preferred concentrations of between about 1 and about 10% H2SO4 or NaOH, preferably between about 2 and about 4% H2SO4 or NaOH) or any combination of the above. The disrupted cells may first undergo a dewatering step to remove some of the water contained in the fermentation broth before being directed to a solvent extraction process. Dewatering may comprise, for example but is not limited to, centrifugation, filtration, evaporation, or combinations thereof. Dewatering may result in a biomass portion containing disrupted cells with a water content less than the water content of the starting fermentation broth. The water content of a dewatered biomass containing disrupted cells and lipids may range from about 10wt% water to about 60wt% water. The disrupted cells may then undergo a solvent extraction process at a ratio between about 100: 1 and about 1 :100 of wet biomass: solvent, or at a ratio of between about 10: 1 to about 1 : 10, or a ratio of about 5: 1 to about 1 :5 or a ratio of about 2: 1 to about 1 :2. A preferred solvent for use at this stage is a solvent with a polarity compatible with the polarity of the lipid fraction and with a sufficiently low boiling point to render solvent removal easier, preferred solvents are short chain alcohol solvents for a highly polar lipid fraction in methanotrophic biomass, e.g. butanol or pentanol or short chain alkane solvents, for microbial non-polar lipids, e.g. hexane or heptane or a combination of polar and nonpolar solvent. Other examples of solvents which can be used in the process of the invention are selected from the group consisting of methanol, ethanol, 1 -propanol, n-butanol, iso-butanol,isoamyl alcohol, 2-methyl-l -butanol, phenethyl alcohol, 1 -pentanol, 1 -hexanol, 1 -heptanol, 1 - octanol, 1 -nonanol, 1 -decanol, tryptophol, isopropanol, 2-butanol, 2-pentanol, 2-hexanol, cyclohexanol, tert-butyl alcohol, tert-amyl alcohol, 2-methyl-2-pentanol, 2-methylhexan-2-ol, 2- methylheptan-2-ol, 3-methyl-3-pentanol, 3-methylactan-3-ol, cyclopentane, cyclohexane, benzene, toluene, diethyl ether, dichloromethane, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, and mixtures thereof. The product from the extraction process is further separated into a light phase, primarily comprising extracted lipids and solvent, and a heavy phase, primarily comprising water, spent biomass, and lipid / solvent carryover. Separation of the light phase and heavy phase may be accomplished by gravimetric methods including but not limited to centrifugation and phase separators. A disk stack centrifuge is employed at the separation stage. The solvent extraction step may be completed at a temperature ranging from about 0°C to about 100°C. In some embodiments of the present invention, the solvent extraction step may be completed at a temperature ranging from about 20°C to about 50°C. The solvent extraction step may be completed at a temperature of about 0°C, about 10°C, about 20°C, about 30°C, about 40°C, about 50°C, about 60°C, about 70°C, about 80°C, about 90°C, or about 100°C. In some embodiments of the present invention, the solvent extraction step may be carried out at a pressure ranging from about 35 kPa (5 psia) to about 13790 kPa (2000psia) or from about 690 kPa (100 psia) to about 6,900 kPa (1000 psia) or from about 6,900 kPa (1000 psia) to about 13,790 kPa (2,000 psia) or from 97 kPa (14 psia) to about 172kPa (25 psia). In some embodiments of the present invention, the solvent extraction step may be completed in a time period ranging from about 1 minute to about 24 hours. In some further embodiments of the present invention, the solvent extraction step may be completed in a time period ranging from about 1 minute to about 60 minutes. In some further embodiments of the present invention, the solvent extraction step may be completed in a time period of about 1 minute, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes. In some further embodiments of the present invention, the solvent extraction step may be completed in a time period ranging from about 1 hour to about 24 hours. In some further embodiments of the present invention, the solvent extraction step may be completed in a time period of about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours,about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.

[0125] “Cl” refers to a one-carbon molecule, for example, CO, CO2, CH4, or CH2O2 or CH3OH. “Cl -oxygenate” refers to a one-carbon molecule that also comprises at least one oxygen atom, for example, CO2, CH3OH, or CH2O2. “Cl -carbon source” refers a one carbon-molecule that serves as a partial or sole carbon source for the microorganism of the disclosure. For example, a Cl- carbon source may comprise one or more of CO, CO2, CH4, CH3OH, or CH2O2. Preferably, the Cl -carbon source comprises CO2 or CH4. A “Cl -fixing microorganism” is a microorganism that has the ability to produce one or more products from a C 1 -carbon source. Often, the microorganism of the disclosure is a Cl -fixing bacterium. In an embodiment, the microorganism of the disclosure is derived from a Cl-fixing microorganism.

[0126] An “autotroph” is a microorganism capable of growing in the absence of organic carbon. Instead, autotrophs use inorganic carbon sources, such as CO and / or CO2. Often, the microorganism of the disclosure is an autotroph. In an embodiment, the microorganism of the disclosure is derived from an autotroph.

[0127] The term “hydrogen-oxidizer” refers to microorganisms that utilize reduced H2 as an electron donor for the production of intracellular reducing equivalents and / or in respiration.

[0128] A “methanotroph” is a microorganism capable of utilizing methane as a sole source of carbon and energy. In certain embodiments, the microorganism of the disclosure is a methanotroph or is derived from a methanotroph. In other embodiments, the microorganism of the disclosure is not a methanotroph or is not derived from a methanotroph.

[0129] The term “knallgas” refers to the mixture of molecular hydrogen and oxygen gas. A “knallgas microorganism” is a microbe that can use hydrogen as an electron donor and oxygen as an electron acceptor in respiration for the generation of intracellular energy carriers such as Adenosine-5 '-triphosphate (ATP).

[0130] The terms “oxyhydrogen” and “oxyhydrogen microorganism” can be used synonymously with “knallgas” and “knallgas microorganism” respectively. Knallgas microorganisms generally use molecular hydrogen by means of hydrogenases, with some of the electrons donated from H2 being utilized for the reduction of NAD+ (and / or other intracellular reducing equivalents) and some of the electrons from H2 being used for aerobic respiration. Knallgas microorganismsgenerally fix C02 autotrophically, through pathways including but not limited to the Calvin Cycle or the reverse citric acid cycle.

[0131] The term “methylotroph” refers to microorganisms that can use reduced one-carbon compounds, such as but not limited to methanol or methane, as a carbon source and / or as an electron donor for their growth.

[0132] A “vessel”, “reaction vessel”, or “column” may be a vessel or container in which one or more gas and liquid streams, or flows may be introduced for bubble generation and / or fine bubble generation, and for subsequent gas-liquid contacting, gas-absorption, biological or chemical reaction, or surface-active material adsorption. In a reaction vessel, the gas and liquid phases may flow in the vertical directions. In a reaction vessel, larger bubbles from a sparger, having a buoyancy force larger than the drag force imparted by the liquid, may rise upwards. Smaller fine bubbles, having a buoyancy force less than or equal to the drag force imparted by the liquid, may flow downward with the liquid, as described by the systems and methods disclosed herein. A column or reaction vessel may not be restricted to any specific embodiment (height to diameter) ratio. A column or reaction vessel may also not be restricted to any specific material and can be constructed from any material suitable to the process such as stainless steel, PVC, carbon steel, or polymeric material. A column or reaction vessel may contain internal components such as one or more static mixers that are common in biological and chemical engineering processing. A reaction vessel may also consist of external or internal heating or cooling elements such as water jackets, heat exchangers, or cooling coils. The reaction vessel may also be in fluid contact with one or more pumps to circulate liquid, bubbles, fine bubbles, and or one or more fluids of the system.

[0133] A “perforated plate” or “plate” may comprise a plate or similar arrangement designed to facilitate the introduction of liquid or additional liquid into the vessel that may be in the form of multiple liquid jets (i.e., accelerated liquid flow). The perforated plate may have a plurality of pores or orifices evenly or unevenly distributed across the plate that allow the flow of liquid from a top of the plate to the bottom of the plate. In some examples, the orifices may be sphericalshaped, rectangular-shaped, hexagonal prism-shaped, conical-shaped, pentagonal prism-shaped, cylindrical-shaped, frustoconical-shaped, or round-shaped. In other examples, the plate may comprise one or more nozzles adapted to generate liquid jets which flow into the column. The plate may also contain channels in any distribution or alignment where such channels are adapted to receive liquid and facilitate flow through into the reaction vessel. The plate may be made ofstainless steel with a predefined number of laser-burnt, machined, or drilled pores or orifices. The specific orifice size may depend upon the required fine bubble size and required liquid, fine bubble, and / or fluid velocities. A specific orifice shape may be required to achieve the proper liquid acceleration and velocity from the plate to break or shear the sparger bubbles into the desired fine bubble size, and to create enough overall fluid downflow to carry the fine bubbles and liquid downward in the reaction vessel. The shape of the orifice may also impact ease of manufacturing and related costs. According to one embodiment, a straight orifice may beemployed due to ease of manufacture.

[0134] The term “controller” includes 1) a measured variable as an input from a source, e.g.., a sensor and transmitter; 2) a calculation device, ex., programmable logic controller (PLC) or a computer, where the setpoint for the measured variable is defined by the user, and calculations are done based on the input from the measured variable; and 3) a manipulated variable as an output, that is used to control the measured variable. The manipulated variable can be a flow control valve, or a motor speed, or an electrical current, or in the case of cascade control, (as in the present disclosure), it can be a setpoint value for another controller. In general, “controller” refers to a single control loop. The term “control system” includes several controllers, all being processed together, as well as possibly other calculations to interpret data.EMBODIMENTS OF THE DISCLOSURE

[0135] Embodiment of this disclosure are described herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the abovedescribed elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0136] Embodiment 1. A method for aerobic gas fermentation comprising providing to a bioreactor comprising a liquid nutrient medium comprising a culture of a microorganism, a gaseous substrate comprising; a first gaseous stream comprising O2 from an O2 source; a second gaseous stream comprising H2 from a H2 source; a third gaseous stream comprising CO2 from a CO2 source; feeding the second and third gaseous streams to the bioreactor at a flow rate toprovide the H2 and CO2 to the bioreactor at a H2: CO2 molar ratio within a predetermined molar ratio range to attain a predetermined value of an uptake molar ratio of H2: CO2 by the microorganism; feeding the first gaseous stream to the bioreactor to provide O2 at a concentration necessary for the microorganism to uptake and ferment with the H2 and CO2 to produce microbial biomass and / or at least one chemical product and an outlet gas stream comprising O2; measuring oxygen concentration in an outlet gas sample stream or outlet gas stream or a headspace above the liquid nutrient medium; transmitting the measured oxygen concentration to a control system which compares the measured oxygen concentration to a predetermined value based on the limiting oxygen concentration (LOC) of hydrogen; and if the oxygen concentration is above or below the predetermined value reducing or increasing the flow rate of the first gaseous stream.

[0137] Embodiment 2. The method of embodiment 1, wherein the second and third gas streams are combined into a blended gas stream and provided to the bioreactor.

[0138] Embodiment 3. The method of embodiment 1 or 2, further comprising introducing a diluent selected from H2, CO2, N2, Ar, or any combination thereof into the bioreactor headspace to dilute the concentration of oxygen in the headspace and maintain it below the predetermined value.

[0139] Embodiment 4. The method of any of embodiments 1 to 3, wherein the outlet gas stream further comprises H2 and CO2.

[0140] Embodiment 5. The method of any of embodiments 1 to 4, wherein the molar ratio of H2:CO2 fed to the bioreactor is in a range of about 7.5: 1 to about 4.5: 1.

[0141] Embodiment 6. The method of any of embodiments 1 to 5, wherein the uptake molar ratio of H2:CO2is in a range of about 6: 1 to about 4: 1.

[0142] Embodiment 7. The method of any of embodiments 1 to 6, wherein the feed rate of the hydrogen in second gaseous stream comprising H2 is from about 5% to about 10% greater than the consumption rate of H2 by the microorganism.

[0143] Embodiment 8. The method of any of embodiments 1 to 7, further comprising compressing the outlet gas stream to generate a compressed outlet gas stream and recycling at least a portion of the compressed outlet gas stream to combine with the second or third gaseous stream or directly into the liquid nutrient medium in the bioreactor.

[0144] Embodiment 9. The method of any of embodiments 1 to 8, further comprising adjusting the flow rate of the second and / or the third gaseous streams or the amount of recycled compressedoutlet gas stream to provide the H2 and CO2 to the bioreactor at a H2: CO2 molar ratio within a predetermined molar ratio range.

[0145] Embodiment 10. The method of any of embodiments 1 to 9, wherein the second gaseous substrate comprising H2 is supplied from a H2 source selected from a water electrolysis process, a hydrocarbon reforming process, a hydrogen purification process, a solid biomass gasification process, a solid waste gasification process, a coal gasification process, a hydrocarbon gasification process, a methane reforming process, a refinery tail gas , a plasma reforming process, a partial oxidation process, or any combination thereof.

[0146] Embodiment 11. The method of any of embodiments 1 to 10, wherein the predetermined value of the O2 concentration in the headspace is from about 1 vol % O2 to about 2 vol % O2.

[0147] Embodiment 12. The method of any of embodiments 1 to 11, wherein the microorganism is a member of genera Cupriavidus.

[0148] Embodiment 13. The method of any of embodiments 1 to 12, further comprising recycling a portion of the liquid nutrient medium from the bottom of the bioreactor to the top of the bioreactor wherein the portion of the liquid nutrient medium is introduced into the bioreactor headspace by at least one spray nozzle.

[0149] Embodiment 14. The method of any of embodiment 1 to 13, further comprising measuring a differential pressure across the spray nozzle, inputting the measured differential pressure to a differential pressure control system and comparing the measured value with a predetermined value and increasing or decreasing the liquid nutrient medium flow rate if the measured differential pressure is below or above a predetermined value.

[0150] Embodiment 15. The method of any of embodiments 1 to 14, wherein if the differential pressure across the spray nozzle shows no or low flow of liquid nutrient medium, shutting off the flow of the first, second and third gaseous streams to the bioreactor.

[0151] Embodiment 16. A method for aerobic gas fermentation comprising providing to a bioreactor comprising a liquid nutrient medium comprising a culture of a microorganism, a gaseous substrate comprising; a first gaseous stream comprising O2 from an O2 source; a second gaseous stream comprising CH4 from a CH4 source; fermenting the CH4 and O2 to produce microbial biomass and / or at least one chemical product and an outlet gas stream comprising O2; feeding the CH4 at a flow rate to maintain the O2 concentration in a headspace above the liquid nutrient medium at a predetermined setpoint below the limiting oxygen concentration (LOC) forCH4; measuring oxygen concentration in an outlet gas sample stream or an outlet gas stream or a bioreactor headspace above the liquid nutrient medium; transmitting the measured oxygen concentration to a control system which compares the measured oxygen concentration to the setpoint; and if the oxygen concentration is above the setpoint, reducing the flow of the first gaseous stream to the bioreactor.

[0152] Embodiment 17. The method of embodimentl6, further comprising recycling a portion of the liquid nutrient medium from the bottom of the bioreactor to the top of the bioreactor wherein the portion of the liquid nutrient medium is introduced into the top of the bioreactor headspace by at least one spray nozzle which distributes the liquid nutrient medium into the headspace.

[0153] Embodiment 18. The method of embodiments 16 or 17, further comprising measuring a differential pressure across the nozzle, inputting the measured differential pressure to a differential pressure control system and comparing the measured value with a predetermined value and increasing or decreasing the liquid nutrient medium flow rate if the measured differential pressure is below or above a predetermined value respectively.

[0154] Embodiment 19. The method of any of embodiments 16 to 18, wherein if the differential pressure across the spray nozzle shows no or low flow of liquid nutrient medium, shutting off the flow of the first and second gaseous streams to the bioreactor.

[0155] Embodiment 20. An apparatus for aerobic gas fermentation comprising a bioreactor vessel comprising an exterior casing having a top and a bottom, the bioreactor vessel containing a liquid nutrient medium comprising a culture, a headspace overlying the liquid; the bioreactor vessel having at least one gas comprising O2 inlet located on the exterior casing and below the top surface of the liquid nutrient medium and in fluid communication with a gas comprising O2 supply conduit in fluid communication with a gas comprising O2 supply unit, the gas comprising O2 supply conduit further comprising a gas comprising O2 flow control valve; the bioreactor vessel having at least one gaseous substrate inlet located on the exterior casing and below the top surface of the liquid nutrient medium and in fluid communication with at least one gaseous substrate supply conduit, the gaseous substrate supply conduit in fluid communication with a gaseous substrate supply unit, the conduit further comprising a gaseous substrate flow control valve; an outlet gas outlet located approximate to the top of the bioreactor vessel, and in fluid communication with at least one outlet gas conduit, and / or an outlet gas sample outlet located approximate to the top of the bioreactor vessel and in fluid communication with at least one outlet gas sample conduit; atleast one oxygen sensor located on the at least one outlet gas conduit or the at least one outlet gas sample conduit or within the headspace; and an O2 control system in electrical communication with the at least one oxygen sensor located in the outlet gas sample conduit and with the optional at least one oxygen sensor located in the headspace of the bioreactor; wherein the outlet gas conduit is in fluid communication with a liquid ring compressor in fluid communication with a compressed outlet gas conduit, wherein the compressed outlet gas conduit is in fluid communication with the gaseous substrate supply conduit or the bioreactor vessel.

[0156] Embodiment 21. The apparatus of embodiment 20, further comprising a liquid nutrient medium recycle loop conduit in fluid communication with the bioreactor vessel and having an inlet approximate to the bottom of the bioreactor vessel and an outlet approximate to the top of the bioreactor vessel; the outlet recycle conduit connected to a spray nozzle located within the bioreactor.

[0157] Embodiment 22. The apparatus of embodiments 20 or 21, further comprising at least one diluent inlet located on the exterior casing and above the top surface of the liquid nutrient medium and in fluid communication with at least one diluent conduit, the diluent conduit in fluid communication with a diluent supply unit.

[0158] Embodiment 23. The apparatus of any of embodiments 20 to 22, further comprising means for measuring a differential pressure across the spray nozzle.

[0159] Embodiment 24. The methods of embodiments 1 and 15 combined.

[0160] Embodiment 25. The methods of any of embodiments 1 to 19 wherein diluent is mixed in the bioreactor headspace by showers of recycled liquid nutrient medium.

Claims

CLAIMS:

1. A method for aerobic gas fermentation comprising: a) providing to a bioreactor comprising a liquid nutrient medium comprising a culture of a microorganism, a gaseous substrate comprising; i) a first gaseous stream comprising O2 from an O2 source; ii) a second gaseous stream comprising H2 from a H2 source; iii) a third gaseous stream comprising CChfrom a CO2 source; b) feeding the second and third gaseous streams to the bioreactor at a flow rate to provide the H2 and CO2 to the bioreactor at a H2: CO2 molar ratio within a predetermined molar ratio range to attain a predetermined value of an uptake molar ratio of H2: CO2 by the microorganism; c) feeding the first gaseous stream to the bioreactor to provide O2 at a concentration necessary for the microorganism to uptake and ferment with the H2 and CO2 to produce microbial biomass and / or at least one chemical product and an outlet gas stream comprising O2; d) measuring oxygen concentration in an outlet gas sample stream or outlet gas stream or a headspace above the liquid nutrient medium; e) transmitting the measured oxygen concentration to a control system which compares the measured oxygen concentration to a predetermined value based on the limiting oxygen concentration (LOC) of hydrogen; and f) if the oxygen concentration is above or below the predetermined value reducing or increasing the flow rate of the first gaseous stream.

2. The method of claim 1, wherein the second and third gas streams are combined into a blended gas stream and provided to the bioreactor.

3. The method of claim 1, further comprising introducing a diluent selected from H2, CO2, N2, Ar or any combination thereof into the bioreactor headspace to dilute the concentration of oxygen in the headspace and maintain the concentration of oxygen below the predetermined value.

4. The method of claim 3, wherein the diluent is mixed in the bioreactor headspace by showers of recycled liquid nutrient medium comprising a culture of a microorganism.

5. The method of claim 1, wherein the molar ratio of H^CChfed to the bioreactor is in a range of about 7.5: 1 to about 4.5: 1.

6. The method of claim 1, wherein the uptake molar ratio of H2:CO2 is in a range of about 6: 1 to about 4: 1.

7. The method of claim 1 wherein the feed rate of the hydrogen in second gaseous stream comprising H2 is from about 5% to about 10% greater than the consumption rate of H2 by the microorganism.

8. The method of claim 4, wherein the outlet gas stream further comprises H2 and CO2. and further comprising compressing the outlet gas stream to generate a compressed outlet gas stream and recycling at least a portion of the compressed outlet gas stream to combine with the second or third gaseous stream or directly into the liquid nutrient medium in the bioreactor.

9. The method of claim 7, further comprising adjusting the flow rate of the second and / or the third gaseous streams or the amount of recycled compressed outlet gas stream to provide the H2 and CO2 to the bioreactor at a H2: CO2 molar ratio within a predetermined molar ratio range.

10. The method of claim 1, wherein the second gaseous substrate comprising H2 is supplied from a H2 source selected from a water electrolysis process, a hydrocarbon reforming process, a hydrogen purification process, a solid biomass gasification process, a solid waste gasification process, a coal gasification process, a hydrocarbon gasification process, a methane reforming process, a refinery tail gas, a plasma reforming process, a partial oxidation process, or any combination thereof.

11. The method of claim 1, wherein the predetermined value of the O2 concentration in the headspace is from about 1 vol % O2 to about 2 vol % O2.

12. The method of claim 1, wherein the microorganism is a member of genera Cupriavidus.

13. The method of claim 1, further comprising recycling a portion of the liquid nutrient medium from the bottom of the bioreactor to the top of the bioreactor wherein the portion of the liquid nutrient medium is introduced into the bioreactor headspace by at least one spray nozzle.

14. The method of claim 13, further comprising measuring a differential pressure across the spray nozzle, inputting the measured differential pressure to a differential pressure controlsystem and comparing the measured value with a predetermined value and increasing or decreasing the liquid nutrient medium flow rate if the measured differential pressure is below or above a predetermined value and shutting off the flow of the first, second and third gaseous streams to the bioreactor if the differential pressure across the spray nozzle shows no or low flow of liquid nutrient medium.

15. A method for aerobic gas fermentation comprising: a) providing to a bioreactor comprising a liquid nutrient medium comprising a culture of a microorganism, a gaseous substrate comprising; i) a first gaseous stream comprising O2 from an O2 source; and ii) a second gaseous stream comprising CH4 from a CH4 source; b) fermenting the CH4 and O2 to produce microbial biomass and / or at least one chemical product and an outlet gas stream comprising O2; c) feeding the CH4 at a flow rate to maintain the O2 concentration in a headspace above the liquid nutrient medium at a predetermined setpoint below the limiting oxygen concentration (LOC) for CH4; d) measuring oxygen concentration in an outlet gas sample stream or an outlet gas stream or a bioreactor headspace above the liquid nutrient medium; e) transmitting the measured oxygen concentration to a control system which compares the measured oxygen concentration to the setpoint; and f) if the oxygen concentration is above the setpoint, reducing the flow of the first gaseous stream to the bioreactor.

16. The method of claim 15, further comprising recycling a portion of the liquid nutrient medium from the bottom of the bioreactor to the top of the bioreactor wherein the portion of the liquid nutrient medium is introduced into the top of the bioreactor headspace by at least one spray nozzle which distributes the liquid nutrient medium into the headspace.

17. The method of claim 16, further comprising measuring a differential pressure across the nozzle, inputting the measured differential pressure to a differential pressure control system and comparing the measured value with a predetermined value and increasing or decreasing the liquid nutrient medium flow rate if the measured differential pressure is below or above a predetermined value respectively and shutting off the flow of the firstand second gaseous streams to the bioreactor if the differential pressure across the spray nozzle shows no or low flow of liquid nutrient medium.

18. The method of claim 17, wherein at least 5 spray nozzles are located and employed in a pattern and direction that enhances gas mixing in the headspace.

19. An apparatus for aerobic gas fermentation comprising: a bioreactor vessel comprising an exterior casing having a top and a bottom, the bioreactor vessel containing a liquid nutrient medium comprising a culture, a headspace above the liquid; the bioreactor vessel having at least one gas comprising O2 inlet located on the exterior casing and below the top surface of the liquid nutrient medium and in fluid communication with a gas comprising O2 supply conduit in fluid communication with a gas comprising O2 supply unit, the gas comprising O2 supply conduit further comprising a gas comprising O2 flow control valve; the bioreactor vessel having at least one gaseous substrate inlet located on the exterior casing and below the top surface of the liquid nutrient medium and in fluid communication with at least one gaseous substrate supply conduit, the gaseous substrate supply conduit in fluid communication with a gaseous substrate supply unit, the conduit further comprising a gaseous substrate flow control valve; an outlet gas outlet located approximate to the top of the bioreactor vessel, and in fluid communication with at least one outlet gas conduit, and an outlet gas sample outlet located approximate to the top of the bioreactor vessel and in fluid communication with at least one outlet gas sample conduit; at least one oxygen sensor located on the at least one outlet gas conduit or the at least one outlet gas sample conduit or within the headspace; and an O2 control system in electrical communication with the at least one oxygen sensor located in the outlet gas conduit, and / or the outlet gas sample conduit and with the optional at least one oxygen sensor located in the headspace of the bioreactor; wherein the outlet gas conduit is in fluid communication with a liquid ring compressor in fluid communication with a compressed outlet gas conduit, wherein the compressed outlet gas conduit is in fluid communication with the gaseous substrate supply conduit or with an inlet on the bioreactor vessel located below the top of the liquid nutrient medium.

20. The apparatus of claim 19 further comprising a liquid nutrient medium recycle loop conduit in fluid communication with the bioreactor vessel and having an inlet approximate to the bottom of the bioreactor vessel and an outlet approximate to the top of the bioreactor vessel; the outlet recycle conduit connected to a spray nozzle located within the bioreactor and or further comprising at least one diluent inlet located on the exterior casing and above the top surface of the liquid nutrient medium and in fluid communication with at least one diluent conduit, the diluent conduit in fluid communication with a diluent supply unit.

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