Method and apparatus for aerobic gas fermentation

Abstract

A control method and apparatus are provided for the safe operation of aerobic gas fermentation by a C1-fixing microorganism to produce various products such as single cell protein or cultured protein. The aerobic fermentation comprises feeding specific gases (one of which is oxygen) into a bioreactor where they're fermented to products such as single cell protein or cultured protein. Specifically, the control apparatus and method involve measuring the O2 concentration in the headspace above a liquid fermentation broth (in a bioreactor) comprising a C1-fixing microorganism and determining if the concentration is at or below a predetermined set point. Using various sensors, flow controllers and transmitters plus a control system, the flow rate of the O2 comprising gas stream into the bioreactor is adjusted or shut off to maintain the O2 concentration in the headspace below the set point.

Description

METHOD AND APPARATUS FOR AEROBIC GAS FERMENTATIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 678, 184 filed on August 01, 2024, and U.S. Provisional Patent Application No. 63 / 790,542 filed on 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 safely operating an aerobic fermentation for production of various products including protein.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, C l -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 flammablemixtures specifically when flammable gases (e.g., hydrogen), are used or produced in the aerobic gas fermentation. The risk of flammability and explosion is a major concern for safe operation of such aerobic gas fermentation processes.

[0006] There is accordingly an ongoing and unmet global need for an improved and safe method and apparatus for safely operating an aerobic gas fermentation process.SUMMARY OF THE INVENTION

[0007] The inventors have found a safe and efficient method and apparatus for operating an aerobic gas fermentation. The disclosed method and apparatus adjust or shut off the flow of gas comprising O2 based on comparison of the measured concentration of O2 in the bioreactor headspace and a predetermined O2 concentration.

[0008] 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 gas 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; a vent gas sample outlet located approximate to the top of the bioreactor vessel and within the gas headspace, and in fluid communication with at least one vent gas sample conduit (or vent gas sample flow conduit) comprising at least one oxygen sensor and optionally at least one oxygen sensor located within the gas headspace, the at least one oxygen sensor in the gas headspace or on the vent gas sample conduit measure a concentration of oxygen in the gas 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.

[0009] In an embodiment, the apparatus further comprises at least one purge gas inlet located above the top surface of the liquid nutrient medium in the gas headspace and in fluid communication with at least one purge gas conduit, the purge gas conduit in fluid communication with a purge gas supply unit, the purge gas conduit further comprising a purge gas shut-off valve. The purge gas supply unit supplies a gas selected from N2, Ar, CO2, or any combination thereof.

[0010] In an embodiment, the apparatus further 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.

[0011] In an embodiment, a safe method for aerobic gas fermentation is also disclosed. The method comprises introducing at least two gaseous feed streams via at least two gaseous feed stream conduits to a bioreactor comprising a liquid nutrient medium comprising a culture of a microorganism, wherein at least one gaseous feed stream comprises a gas comprising O2, and at least one feed stream comprises a gaseous substrate selected from a gas comprising H2, CO2, CH4 or combination thereof, and fermenting the gaseous substrate to produce microbial biomass and / or at least one chemical product and a tail gas which forms a headspace above the liquid nutrient medium in the bioreactor; measuring gaseous oxygen concentration in the headspace of the bioreactor; transmitting the oxygen concentration to a control system which determines whether the measured concentration is above a first set point or rate of change of O2 concentration is above a predetermined value and sends a signal to an O2 flow control valve located on the gas comprising O2 conduit to reduce the flow of the gas comprising O2 feed stream.

[0012] In an embodiment, the method further comprises flowing a purge gas into the headspace of the bioreactor when the second set point is above 2.5 vol % O2. The purge gas is selected from Ar, N2, CO2 or any combination thereof.

[0013] In an embodiment, the method further comprises introducing a diluent gas stream into the gas headspace to dilute the concentration of O2 in the gas headspace. The diluent gas stream is selected from H2, CO2 N2. Ar. CO2, or any combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Fig. 1 illustrates an apparatus in accordance with an embodiment.

[0015] Fig. 2 presents several plots showing headspace O2 concentration and dissolved O2 concentration as a function of time.DETAILED DESCRIPTION

[0016] Embodiments 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 subjectmater recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0017] The inventors have found a safe and efficient method and apparatus for safely operating an aerobic gas fermentation process. The disclosed method and apparatus adjust or shut off the flow of gas comprising O2, based on comparison of the measured concentration of O2 in the bioreactor headspace and a predetermined O2 concentration or rate of change of O2 concentration. Such comparison is carried out by a control system.

[0018] In an embodiment, the apparatus comprises a bioreactor vessel comprising an exterior casing having a top and a botom, the bioreactor vessel containing a liquid nutrient medium comprising a culture, a gas 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; a vent gas sample outlet located approximate to the top of the bioreactor vessel and within the gas headspace, and in fluid communication with at least one vent gas sample conduit comprising at least one oxygen sensor and optionally at least one oxygen sensor located within the gas headspace, the at least one oxygen sensor in the gas headspace or on the vent gas sample conduit measure a concentration of oxygen in the gas 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. The bioreactor vessel further comprises one or more gaseous substrate sensors for measuring the concentration of gaseous substrate in the culture, and in communication with flow controller located on the gaseous substrate supply conduit. The concentration of the gaseous substrate in the liquid nutrient medium is measured and the gaseous substrate flow rate into the bioreactor is adjusted based on the concentration of the gaseous substrate.

[0019] In an embodiment, the apparatus further comprises at least one purge gas inlet located above the top surface of the liquid nutrient medium in the gas headspace and in fluidcommunication with at least one purge gas supply conduit, the conduit in fluid communication with a purge gas supply unit, the conduit further comprising a purge gas shut off valve. The control system controls the flow of purge gas from the purge gas supply unit to the bioreactor.

[0020] In an embodiment, the gaseous substrate supply unit further comprises two supply units; a first gaseous substrate supply unit which is in communication with a first gaseous substrate supply conduit and is in communication with the at least one gaseous substrate inlet on the bioreactor vessel; and a second gaseous substrate supply unit supplying a second gaseous which is in communication with a second gaseous substrate supply conduit in communication with a second gaseous substrate inlet on the bioreactor vessel located on the exterior casing and below the surface of the liquid nutrient medium.

[0021] In specific embodiment, as illustrated in Fig. 1. the gaseous substrate supply unit is split into two separate units such that a first unit supplies a gas comprising H2 as the first gaseous substrate and the second supply unit supplies a second substrate which is a gas comprising CO2.

[0022] In another embodiment, when the gaseous substrate is CFU and only one gaseous substrate supply unit is provided along with the corresponding conduit, CFU sensor, flow control valve, shut off valve and gaseous substrate inlet to the vessel.

[0023] In an embodiment, the bioreactor vessel further 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 a gas 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.

[0024] In an embodiment, a safe method for aerobic gas fermentation is also disclosed. The method comprises introducing at least two gaseous feed streams via at least two gaseous feed stream conduits to a bioreactor comprising a liquid nutrient medium comprising a culture of at least one microorganism, wherein at least one gaseous feed stream comprises a gas comprising O2, and at least one feed stream comprises a gaseous substrate selected from a gas comprising H2, CO2. CFU or combination thereof, and fermenting the gaseous substrate to produce microbial biomass and / or at least one chemical product and a tail gas which forms a headspace above the liquid nutrient medium in the bioreactor; measuring gaseous oxygen concentration in the headspace of the bioreactor; transmitting the oxygen concentration to a control system which determines whether the measured concentration is above a first set point or rate of change of O2 concentration is above a predetermined value and sends a signal to an O2 flow control valve located on the gas comprising O2 conduit to reduce the flow of the gas comprisingO2 feed stream. In an embodiment, the rate of change of O2 concentration is in a range of about 0. 1 vol % to about 2 vol % per minute. By way of an example, if the rate of change of O2 concentration is about 0.5 vol % per minute the control system sends a signal to an O2 flow control valve located on the gas comprising O2 conduit to reduce the flow of the gas comprising O2 feed stream. It is understood that the rate of change of the O2 concentration which triggers the control system to adjust the flow of the gas comprising O2 will depend on parameters such as the size of the bioreactor vessel, the frequency of the measurements, lag time between measurement and when the measurement is received by the control system. By way of an example, the intended setpoint flow for H2 and CO2 is 85 vol %.

[0025] In an embodiment, the method further comprises flowing a purge gas into the headspace of the bioreactor when the second set point is point above 1% vol% O2 to about 2.75 vol % O2 or from about 1.5 vol. % to about 2.5vol. % O2 or above 2.5 vol % O2 The purge gas is selected from Ar, N2, CO2 or any combination thereof.

[0026] In an embodiment, the method further comprises introducing a diluent gas stream continuously into the gas headspace to dilute the concentration of O2 in the gas headspace. The diluent gas stream is selected from H2, CO2 N2, Ar, or any combination thereof.

[0027] In an embodiment, the method comprises separately introducing at least two feed streams to a bioreactor comprising a culture of a microorganism in a liquid nutrient medium, wherein at least one feed stream comprises a gas comprising O2 and at least one feed stream comprises a gaseous substrate selected from a gas comprising H2. a gas comprising CO2. a gas comprising CH4 or any combination thereof.Definitions

[0028] Unless otherwise defined, the following terms as used throughout this specification are defined as follows:

[0029] A “microorganism’’ is a microscopic organism, especially a bacterium, archaeon, virus, or fungus. In an embodiment, the microorganism of the disclosure is a bacterium.

[0030] 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.

[0031] 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 “geneticallyengineered” 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.

[0032] “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.

[0033] “Heterologous” refers to a nucleic acid or protein that is not present in the wild-ty pe 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.

[0034] 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, Mycobacter aides, 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 Symbiobacterium.

[0035] 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.

[0036] “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.

[0037] “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 entirety 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, a mutation 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.

[0038] 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 wild-type 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 enzy mes 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.

[0039] 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, (defined below) 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. 11220700 B2 provides a process for production of at least one product from a methane- containing feedstock. The process comprises providing a gaseous substrate comprising CPU 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.

[0040] “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, CH?OH, or CH2O2. Preferably, the Cl-carbon source comprises CO2 or CP . A '‘Cl -fixing microorganism” is a microorganism that has the ability to produce one or more products from a Cl-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.

[0041] 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.

[0042] The term "hydrogen-oxidizef ’ refers to microorganisms that utilize reduced H2 as an electron donor for the production of intracellular reducing equivalents and / or in respiration.

[0043] A “methanotroph” is a microorganism capable of utilizing methane as a sole source of carbon and energy7. 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.

[0044] 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).

[0045] 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 microorganisms generally fix CO2 autotrophically, through pathways including but not limited to the Calvin Cycle or the reverse citric acid cycle.

[0046] 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 grow th.

[0047] 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 liquidphases 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.

[0048] 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 spherical-shaped, 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 of stainless 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 be optimal due to ease of manufacture.

[0049] 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, andcalculations 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.Microorganisms for use in the disclosed method

[0050] 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.

[0051] 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 Rcdstonia. 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 Cupriavidus 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 .

[0052] 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.

[0053] 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.

[0054] 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 w ater vapor, rises to the headspace of the bioreactor.

[0055] 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.

[0056] 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.

[0057] In one embodiment, the aerobic bacteria may produce a product such as acetone, isopropanol, 3-hydroxyisovaleryl-CoA, 3-hydroxyisovalerate, isobutylene, isopentenyl pyrophosphate, dimethylallyl pyrophosphate, isoprene, famesene, 3-hydroxybutyryl-CoA, crotonyl-CoA, 3 -hydroxy butyrate, 3-hydroxybutyiylaldehyde, 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.

[0058] 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, chorismate-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 (SAF) 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

[0059] “Substrate” refers to a carbon and / or energy source for the microorganism of the disclosure. Often, the substrate is gaseous and comprises a Cl-carbon source, for example, CO, CO2, CH3OH, CH2O2 and / or CH4. The substrate may further comprise an energy source such as H2.

[0060] 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 1 vol. % to about 100 vol. %. By way of an example, the ratio of IfeCChiCh fed to the microorganism is optimally 6:1 :2. 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.

[0061] 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 oxygen-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.

[0062] The gaseous substrate may also comprise CO2. For example, the gaseous substrate may comprise about 1 vol %to about 100 vol % CO2.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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 Ci- carbon source may be captured from the industrial process before it is emitted into the atmosphere, using any known method.

[0070] 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 biomass may include lignocellulosic material and may also include microbial biomass. Lignocellulosic material may include agriculture w aste and forest waste.

[0071] 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.

[0072] “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.

[0073] 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., non-dried) 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.

[0074] 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.

[0075] 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).

[0076] 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.

[0077] "‘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.

[0078] 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, 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

[0079] 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.

[0080] 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. As used herein, the terms “culture” and “fermentation” areused interchangeably. These terms encompass both the grow th phase and product biosynthesis phase of the culture / fermentation process.

[0081] 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 well known in the art.

[0082] 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 ensure that gas in the liquid phase does not become limiting, and maximum product concentrations to avoid product inhibition. In particular, the rate of introduction of the substrate may be controlled to ensure that the concentration of gas in the liquid phase does not become limiting.

[0083] 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 gas conversion 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 optimum 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.

[0084] 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 readingthis 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.

[0085] 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.

[0086] 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 optimal 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.

[0087] 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,

[0088] Fig. 1 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. Dotted / dashed lines depict transfer of signals from the sensors to the control system, flow controller valves, shut-off valves, and flow transmitters. Individual gases, gas comprising O2. gas comprising H2, and gas comprising CO2 or CH4 enter 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.

[0089] A gas comprising O2 is provided by gas supply unit (10) in fluid communication with a gas comprising O2 supply conduit (18) which delivers the gas comprising O2 to bioreactorvessel (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. Each inlet is connected to a sparger (not shown) on the inside of the vessel.

[0090] A "sparger" may comprise a device to introduce gas into a liquid, injected as bubbles, to agitate it or to dissolve the gas in the liquid. Example spargers may include orifice spargers, sintered spargers, and drilled pipe spargers. In certain configurations drilled pipe spargers may be mounted horizontally. In other examples, spargers may be mounted vertically or horizontally. In some examples, the sparger may be a perforated plate or ring, sintered glass, sintered steel, porous rubber pipe, porous metal pipe, porous ceramic or stainless steel, drilled pipe, stainless steel drilled pipe, polymeric drilled pipe, etc. The sparger may be of various grades (porosities) or may include certain sized orifices to produce a specific sized bubble or range of bubble sizes.

[0091] The supply conduit (18) also comprises a gas comprising O2 shut-off actuated valve (12), and a gas comprising O2 flow controller (36). The supply conduit (18) further comprises at least one oxygen sensor (01) to measure the concentration of oxygen in the stream flowing through the conduit. The gas comprising O2 supply conduit (18) further comprises at least one gas comprising O2 control valve (114) and at least one gas comprising O2 flow transmitter (24). In an embodiment, the supply conduit (18) is split into two or more lines which are connected at various point vertically from the bottom of the external casing to near the top of the liquid fermentation broth.

[0092] As shown in Fig. 1, in one embodiment where the gaseous substrate comprises a gas comprising 142 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 gas comprising H2 inlet is connected to a sparger (not shown) on the inside of the vessel. The supply conduit (32) comprises a gas comprising H2 shut-off actuated valve (14), and a gas comprising H2 flow controller (38). The supply conduit (32) further comprises at least one H2 sensor (03) to measure the concentration of hydrogen flowing through the conduit. The gas comprising 142 supply conduit (32) further comprises at least one gas comprising H2 control valve (122) and at least one gas comprising H2 flow transmitter (26).

[0093] As shown in Fig. 1, 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 communicationwith 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 gas comprising CO2 inlet is connected to a sparger (not shown) on the inside of the vessel. The gas comprising CO2 supply conduit (22) comprises a gas comprising CO2 shut-off actuated valve (16), and a gas comprising CO2 flow controller (40). The supply conduit (22) further comprises at least one CO2 sensor (05) which measures the concentration of the CO2 flowing through the conduit. The gas comprising CO2 supply conduit (22) further comprises at least one gas comprising CO2 control valve (122) and at least one gas comprising CO2 flow transmitter (28).

[0094] In an alternate embodiment, the gas comprising H2 and gas comprising CO2 can be supplied to the bioreactor using only supply unit (20 or 30), 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 (38 or 40), one flow transmitter (26 or 28). However, sensors for H2 (03) and CO2 (05) would be provided on the chosen conduit.

[0095] 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 (and one sparger), one CH4 flow control valve (112 or 122), one CH4 flow controller (38 or 40), one CH4 flow transmitter (26 or 28), and one CH4 sensor (03 or 05).

[0096] In another embodiment the bioreactor vessel further comprises a purge gas supply unit (200) in fluid communication with a purge gas supply conduit which delivers the purge gas to the bioreactor vessel (100) through an inlet located above the top surface of the liquid nutrient medium in the gas headspace. The purge gas inlet is connected to a sparger on the inside of the vessel. The supply conduit comprises a purge gas shut-off actuated valve (210). The purge gas supply unit supplies a purge gas comprising an inert gas selected from N2, Ar, CO2, or any combination thereof. Purge gas is supplied at an appropriate or desired pressure and flow is monitored to ensure appropriate flow / pressure within the conduit supplying the purge gas to the headspace of the bioreactor.

[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 diluent a gas selected from H2, CO2 N2, Ar, or any combination thereof. The diluent is continuously fed to the headspace of the bioreactor as a safety measure to dilute the gases produced by the fermentation or passed through the fermentation, e.g. not reacted.

[0098] In an embodiment, purge gas supply unit and diluent supply unit are not separate (not shown in the Figure); and the conduit supplying the diluent and purge gas is split into two conduits for supplying diluent and purge gas separately to the bioreactor vessel. In this case, the diluent supply conduit is split before or upstream of the gas shut-off actuated valve (210).

[0099] The bioreactor (100) contains a liquid culture medium and a gas headspace from above the top of the liquid culture medium to the top of the vessel. The bioreactor vessel further comprises one or more oxygen sensor (68, 70) within the liquid culture which measures the concentration of dissolved oxygen in the culture and is in communication with the at least one gas comprising O2 flow controller (36) through the gas comprising O2 flow transmitter (24). The bioreactor vessel (100) comprises a vent gas sample flow conduit (300) having at least one oxygen sensor but typically redundant sensors, for example sensors (72), (74), and (76). Although Fig. 1 shows the use of three oxygen sensors, the description which follows will describe the case of one oxygen sensor for simplicity, but it is to be understood that multiple sensors can be used. The vent gas sample also has at least two vent gas sample flow meters / transmitters. One flow meter / transmitter, e.g. (92) can be used for controlling the flow while another flow meter / transmitter. e.g. (94) only monitors the gas flow. The reason for having at least two flow meters is that if the flow meter used for control fails, the monitor can detect that sample flow is lost and safely shut down the system preventatively.

[0100] In order to ensure that the safety system functions within its given parameters, especially quickly responding to O2 concentration changes or rate of changes in the head space of the bioreactor, there should be minimal delay in measuring and transmitting the vent gas sample flow rate to the flow controller (110) or the control system. The flow controller (110) operates by adjusting flow to ensure the flow in the vent sample gas conduit is above a predetermined set point. If inadequate flow for proper sampling is detected on the gas sample conduit by the independent flow meter / transmitter, e.g., (92), an interlock signal (80) is sent from e.g., flow transmitter (92) the interlock (80) will shut off the oxygen flow to the bioreactor by closing the gas comprising O2 shut-off actuated valve (12) and O2 gas control valve (114). Additionally, the gaseous substrate flow into the bioreactor will also be shut off by closing shut off valve(s) (14) and / or (16) as well as flow control valve(s) (112) and / or (122).

[0101] The oxygen sensor (72) on the vent 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 gas comprising O2 flow controller (36). In another embodiment the vent gas sample has multiple oxygen sensors. The concentration of the O2 measured by the sensors is averaged by the control system and this value is used to adjust theflow of gas comprising O2 into the bioreactor using the flow controller (36) on the gas comprising O2 supply conduit.

[0102] In an embodiment, vent gas sample flow conduit (300) is split into two or more separate conduits each having at least one oxygen sensor for measuring the concentration of oxygen gas in the gas headspace and are in communication with the gas comprising O2 flow controller. Additionally, each vent gas sample conduit has two flow meters as described above. This embodiment uses two or more vent gas sample conduits for providing redundancy in the safety system described herein. Thus, flow in the two vent gas sample conduits is measured and averaged. However, in the case where one flow meter / transmitter shows no flow or low flow or a flow vastly different from what it previously measured and different from the flow measured by the other meter in the other conduit, then an interlock signal (80) is sent from e.g., flow transmitter (92) to controller (50) which removes the sensor(s) with inadequate flow or erroneous flow from the averaging function and only the other flow meter and associated O2 sensor on the other conduit is used for measurements. When two or more conduits are used, multiple oxygen sensors can also be used in each conduit. Whether using multiple O2 sensors in each of the two or more conduits, the O2 concentration from the multiple conduits is averaged and the value is used for controlling the flow of the gas comprising O2 to the bioreactor. By using more than one vent sample conduit, if one O2 sensor on one conduit fails, then the sensors on the other vent gas sample conduits (or the redundant sensors on the same conduit) can be used to control the flow of the gas comprising Chto the bioreactor. If the flow is blocked or the flow controller fails on one conduit, the sensors in the other conduits can be used to control the flow of gas comprising O2 to the bioreactor. This embodiment provides a fail-safe system even in the event that three out of four sensors fail.

[0103] The flow controller (110) operates by adjusting flow to ensure the flow in the vent sample gas conduits is above a predetermined set point. The predetermined setpoint is based on the amount of time required for the gas to travel from the bioreactor headspace to the sensor, which is a function of the geometry of the sample conduit. The flow setpoint is configured to ensure that the vent gas sample is not more than a predetermined age. The predetermined age of the vent gas sample is less than the minimum time it would take for a flammable gas composition to develop, in order to be able to respond before a hazardous condition exists. The minimum time for the hazardous condition to exist or develop is a function of various parameters and is determined for each system independently. In order to prevent a hazardous condition to develop, the age of the vent gas sample varies from about 5 seconds to about 1 minutes. In some embodiments the age of the vent gas sample can be less than 5 seconds.

[0104] 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. The oxygen sensor in the headspace is in communication with the at least one gas comprising O2 flow controller. No flow monitoring is required for O2 sensor placed in the headspace of the bioreactor. O2 sensors placed in the headspace have the same function as that of the sensors placed on the vent gas sample flow conduit, however sensors placed on the vent gas sample flow conduit are more protected against water ingress and thus more reliable.

[0105] In an embodiment, when the gaseous substrate is the gas comprising CH4 at least one of the vent gas sample conduits 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 vent gas sample conduit measure concentration of CH4 in the gas headspace and are in communication with the control system.

[0106] The bioreactor of the disclosure comprises a control system. 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 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.

[0107] Referring to Fig. 1, the inner controller is the O2 flow controller (control block 36) which receives a flow setpoint from the outer controller. The outer controller can be selected from two different sources. These two sources consist of a dissolved oxygen controller (control block 56), which adjusts the oxygen flow setpoint in order to maintain a particularconcentration of dissolved oxygen, as measured by dissolved oxygen sensors (sensors 68 and 70), and a headspace oxygen controller (control block 50) which maintains a particular concentration of gaseous oxygen in the bioreactor headspace as measured by oxygen sensors (72), (74), and (76) in the vent gas sample conduit. The oxygen control selector (control block 44) selects which source gives the feedback to the oxygen flow controller (36). The source selection will automatically transition from the dissolved oxygen (56) controller to the headspace oxygen controller (50) if the oxygen concentration in the headspace rises above a threshold value. In some embodiments, the source selector can also automatically transition back from the headspace oxygen controller to the dissolved oxygen controller, if the dissolved oxy gen concentration exceeds a threshold value.

[0108] 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 the transmitters send the concentration or flow signals via cables 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 system or individual controllers control the flow of purge gas from the purge gas supply unit to the bioreactor or the flow of diluent gas from the diluent gas supply unit to the bioreactor..

[0109] As illustrated in Fig. 1. each of H2. O2 and Cl containing substrate (i.e. CO2) flow lines have a standard flow controller which uses a control valve to adjust the flow to match a setpoint value, based on a feedback signal from a flow transmitter. This combination of controller, valve and transmitter correspond to the following items in Fig. 1:• for O2. controller 36, valve 114. flow transmitter 24.• for H2, controller 38, valve 122, flow transmitter 26.• for Cl containing substrate, controller 40. valve 112, flow transmitter 28.

[0110] The Bioreactor gas flow control for H2 and C 1 containing substrate is based on the ratio of H2 to Cl containing substrate which is set manually by the operator based on the stoichiometry of reaction and can be refined based on observation of the tail gas composition. [OlH] The flow control setpoint for each of H2 (control block 38) and Cl containing substrate (control block 40) is calculated based on the manually input ratio between H2 and C 1 containing substrate, and the total gas flow Set Point. In some embodiments, this total flow setpoint may have the dynamic O2 gas setpoint (control block 36) subtracted from it before applying the ratioof H2 to Cl containing substrate to the remainder of the flow. Splitting of this total gas flow into individual setpoints for H2 and Cl containing substrate flow is accomplished by the total gas flow ratio controller (control block 42).

[0112] Gas flow control for O2 can be a cascade controller, with the inner controller being the O2 flow controller (control block 36) receiving a flow setpoint from the outer controller. The outer controller can be selected between two different sources. These two sources consist of a dissolved oxygen controller (control block 56), which adjusts the oxygen flow setpoint in order to maintain a particular concentration of dissolved oxygen, as measured by dissolved oxygen sensors (sensors 68 and 70), and a headspace oxygen controller (control block 50) which maintains a particular concentration of gaseous oxygen in the bioreactor headspace. The oxygen control selector (control block 44) selects which source gives that feedback to the oxygen flow controller (36). The source selection will automatically transition from the dissolved oxygen (56) controller to the headspace oxygen controller (50) if the oxygen concentration in the headspace rises above a threshold value. In some embodiments, the source selector can also automatically transition back from the headspace oxygen controller to the dissolved oxygen controller, if the dissolved oxygen concentration exceeds a threshold value.

[0113] In the case of both the dissolved oxygen and headspace oxygen measurements, multiple redundant sensors are used for safe operation, in case of a deviation or a drift in a single sensor. For the dissolved oxygen, these are dissolved oxygen sensors (68 and 70). For the headspace oxygen, these are oxygen sensors (72. 74 and 76).

[0114] In case of a deviation in oxygen concentrations that is not resolved rapidly enough by the controllers, there is also an interlock system to shut off oxygen to the system if a high headspace oxygen concentration is detected. This interlock (08) occurs when two of the three (or in some embodiments, one of the three) headspace oxygen sensors (72, 74, 76) read above a threshold headspace oxygen value, and the interlock action shuts off the inlet oxygen at the oxygen shutoff valve (12) and optionally starts flow of purge gas. The oxygen control valve (114) will also be closed by the interlock action.

[0115] In addition to redundant measurements of the headspace oxygen, it is also important to verify that these measurements are occurring with an appropriate frequency to detect a rise in headspace oxygen before it reaches a dangerous level. The vent gas sample line is configured with a flow controller (110) to ensure the sample flow is fast enough to have an acceptable delay in the measurement. As a precaution, if this flow is measured to be too low, an independent flow monitor (92) will activate an interlock to shut off the inlet oxygen at theoxygen shutoff valve (12). The oxygen control valve (114) will also be closed by the interlock action.

[0116] Another possible cause of high oxygen in the bioreactor headspace is the composition of the individual feed gases. The gas is fed to the bioreactor with a much higher inlet oxygen content than what is seen in the head space, and its concentration is reduced significantly by reaction with the other gases. If oxygen concentration in the gas comprising O2 inlet increases significantly over a short period of time, the bioreactor can very quickly have higher than expected oxygen in the headspace, with the potential of a flammable mixture. Similarly, if the H2 or Cl containing substrate concentration drops significantly in those streams, there may not be enough of either Cl containing substrate or H2 to react with the oxygen, and the unreacted oxygen will raise the concentration in the head space. To avoid this issue, each gas feed is equipped with a sensor to monitor composition. If the O2 concentration measured by the O2 inlet gas sensor (01) has a high rate of change of increase in concentration, it will trigger an interlock (02). A low rate of change in 02 concentration can be safely handled by ordinary operation of the oxygen controls. If the H2 concentration measured by the H2 inlet gas sensor (03) is lower than expected, it will trigger an interlock (04). If the CO2 concentration measured by the CO2 inlet gas sensor (05) is lower than expected, it will trigger an interlock (06). All three of these interlocks (02, 04, 06) will shut off the inlet oxygen at the oxygen shutoff valve (12). The oxygen control valve (114) will also be closed by the interlock action.

[0117] Similarly to the composition, the flow rate of the gases not meeting their intended setpoints can also cause high oxygen in the bioreactor headspace. Inadequate flow of either gas comprising Cl containing substrate or H2 to react with the oxygen will cause the unreacted concentration in the head space. To avoid this issue, each gas flow controller is tied to an interlock for low flow. If the H2 flow measured by the H2 flow controller (38) is lower than setpoint by a defined proportion for a set time, it will trigger an interlock (09). If the Cl containing substrate flow measured by the gas comprising C l containing substrate flow controller (40) is low er than setpoint by a defined proportion for a set time, it will trigger an interlock (10). Both of said interlocks (09, 10) will shut off the inlet oxygen at the oxygen shutoff valve (12). The oxygen control valve (114) will also be closed by the interlock action. The set point flow rate is defined as the target flow rate. Low flow rate is then defined as 80% (or lower) of the set point flow rate. It should be pointed out that the set point flow' rate can vary during the process and is set by the operator. At the start of the process, the inlet gaseous feed streams flow rates are low. The inlet gaseous feed streams flow rates are increased over a period of time based on the microorganism growth rate. The final inlet gaseous feed streamsflow rates are those when steady state has been achieved. In addition to microorganism grow th rate, the set point flow rate for feed gases also depends on several other parameters including but not limited to availability of the gaseous stream components (or design gas flow rate), utilization of the gases by the microorganism, portion of the tail gas being recycled, flow rate of diluent gas, etc. In view of the changes in these process parameters, the set point flow rate of the feed gases need to be adjusted otherwise the flow sensors will detect a low flow rate causing the control system to shut off the gaseous substrate flow unnecessarily.

[0118] Hydrogen is highly flammable, and even small amounts 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 %. Limiting Oxygen Concentration for methanol is approximately 10 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, a set point is defined based on LOC as per NFPA guidelines. As stated above, the set point depends on the t pe of flammable gaseous substrate used. In general, oxygen is flammable at about 4.755 vol % when hydrogen is more than 3-4 vol % in the gas headspace. Therefore, when hydrogen is present as the gaseous substrate, a first set point for O2 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. When CH4 is used as a gaseous substrate and no hydrogen is used, the first set point is in a range from about 5 vol % to 15 vol % of oxygen or about 6 vol % to 15 vol % or about 7 vol % to 15 vol % or about 8 vol % to 15 vol % or about 9 vol % to 15 vol % or about 10 vol % to 15 vol % or about 11 vol % to 15 vol % or about 12 vol % to 15 vol % or about 13 vol % to 15 vol % or about 14 vol % to 15 vol % of oxygen. Set points also depend on the type of reactor vessel, scale of production, calculated response time of the system, etc. In general, safety guidelines provided by American National Fire Protection Association (NFPA) are considered while determining set point.

[0119] If the oxygen concentration in the headspace rises above a first set point, flow of the gas comprising O2 is reduced. In an embodiment, if the oxygen concentration in the headspace rises above the first set point, the flow of gas comprising oxygen is reduced by 20 %. After this reduction, if the oxygen concentration falls below the first set point, the process returns to normal operation, with the controller continuing to adjust flow starting from this reduced flow set point. The purpose of this set point is to rapidly constrain a deviation in oxygen concentration without triggering a full shut off of the gases and purging the headspace. Thefirst set point has a trip timer, where the set point needs to be exceeded for a given duration before the interlock is triggered. This prevents nuisance trips from a single bad reading from the sensors or any measurement noise to occur.

[0120] If the flow of the gas comprising O2 is reduced as in the embodiment above and the O2 concentration continues to rise in the headspace above a second setpoint, or the oxygen concentration in the headspace is initially above a second set point, the second set point having a higher value than the first set point, an interlock action is initiated whereby the flow of the gas comprising O2 and the flow of the gaseous substrate(s) into the bioreactor is shut off and a purge gas is flowed into the headspace of the bioreactor vessel to rapidly bring the oxygen concentration back to “safe"’ levels. The interlock actions, which are defined in the control system logic, and the control system then sets the controller to close the shut off valve of the gas comprising O2 and gaseous substrate(s). The flow control valves on the individual flow controllers are also shut. The control system also opens the actuated valve for the purge. The purge gas continues to flow, either until the oxygen concentration in the headspace is measured below a safe level, or a predetermined amount of time has elapsed for the purge which would ensure a safe composition in the headspace. The second set point also has a trip timer, where the set point needs to be exceeded for a given duration before the interlock is triggered. This prevents nuisance trips from a single bad reading from the sensors or any measurement noise to occur.

[0121] In an embodiment, when the gaseous substrate is a gas comprising H2, the second set point is greater than 1 vol % O2 to about 2.75 vol % O2 or about 1 .25 vol % O2 to about 2.75 vol % O2 about 1.5 vol % O2 to about 2.75 vol % O2 or about 1.75 vol % O2 to about 2.75 vol % O2 or about 2 vol % O2 to about 2.75 vol % O2 or about 2,25 vol % O2 to about 2.75 vol % O2 or about 2.5 vol % O2 to about 2.75 vol % O2.

[0122] In an embodiment, when the gaseous substrate is a gas comprising CH4, the second set point is in the range from about 6 vol % O2 to about 18 vol % O2 or about 7 vol % O2 to about 18 vol % O2 or about 8 vol % O2 to about 18 vol % O2 or about 9 vol % O2 to about 18 vol % O2 or about 10 vol % O2 to about 18 vol % O2 or about 11 vol % O2 to about 18 vol % O2 or about 12 vol % O2 to about 18 vol % O2 or about 13 vol % O2 to about 18 vol % O2 or about 14 vol % O2 to about 18 vol % O2 or about 15 vol % O2 to about 18 vol % O2 or about 16 vol % O2 to about 18 vol % O2 or about 17 vol % O2 to about 18 vol % O2.

[0123] 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 fdtration), 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 tipbased 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.

[0124] 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.

[0125] 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

[0126] 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.

[0127] In some embodiments, the microbial biomass has a unit value. In one embodiment, the microbial biomass has a market value.

[0128] 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, an acknowledgement that that prior art forms part of the common general knowledge in the field of endeavour in any country.

[0129] 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.

[0130] 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.

[0131] 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 otherwiseclaimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.EXAMPLE

[0132] A fermentation broth comprising Cupriavidus necator microorganism in a liquid nutrient was fed air, H2, and CO2 thereby producing biomass. However, a process upset started to affect the culture. Fig. 2 provides several plots showing how the process and apparatus of the disclosure were able to handle this upset. In the graph of Fig. 2, the x-axis represents time in minutes, the lefty-axis represents percent O2 in the headspace above the fermentation broth and mg / L of dissolved O2 in the broth. The right y-axis represents Normal Liters Per Minute (NLPM) of air being flowed into the fermentation broth. NLPM is a unit of flow rate that standardizes the measurement of gas flow (liters) to a set of conditions, typically 0°C (273. 15 K) and 1 atmosphere (101.325 kPa or 760 mm Hg). The plots in Fig. 2, show that at approximately 22 minutes into the experiment as a result of this upset, the dissolved O2 (DO) concentration began increasing toward its set point (SP) of 0.4 mg / 1 followed by the headspace O2 concentration increasing above its SP of 2%. In response to this upset, the control system initiated a reduction in airflow, causing the DO concentration to a decrease below its SP and the headspace O2 concentration to flatten out and also decrease below its SP. Subsequent spikes in dissolved oxygen above the SP, resulted in the control system further reducing the inlet air flow which resulted in further reductions in headspace O2 concentration. The control system continued to operate effectively until the cause of the problem was determined and fixed. This is shown by the last spike in the plot of DO concentration versus time decreasing versus the previous spike. This example shows that the control system and process of the disclosure were able to effectively control headspace O2 concentration and maintained the concentration below the SP thus operating the process safely.EMBODIMENTS OF THE DISCLOSURE

[0133] Embodiment 1. An apparatus for aerobic gas fermentation, 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 gas 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 exteriorcasing 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; a vent gas sample outlet located approximate to the top of the bioreactor vessel and within the gas headspace, and in fluid communication with at least one vent gas sample conduit comprising at least one oxygen sensor and optionally at least one oxygen sensor located within the gas headspace, the at least one oxygen sensor in the gas headspace or on the vent gas sample conduit measure a concentration of oxygen in the gas 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.

[0134] Embodiment 2. The apparatus of embodiment 1, wherein the at least one vent gas sample conduit further comprises at least one vent gas sample flow' monitors and / or transmitters for monitoring the flow of the vent gas in the vent gas sample conduit.

[0135] Embodiment 3. The apparatus of embodiments 1 or 2, wherein the gaseous substrate supply unit supplies a gaseous substrate selected from a gas comprising H2, a gas comprising CO2, a gas comprising CEE or any combination thereof.

[0136] Embodiment 4. The apparatus of any of embodiment 1 to 3, wherein the gas comprising O2 supply conduit further comprises a) an 02 gas sensor to monitor the O2 concentration and transmits the O2 concentration to the control system; b) a gas comprising O2 shut-off actuated valve in communication with the control system; and c) a gas comprising O2 flow control valve in communication with the control system.

[0137] Embodiment 5. The apparatus of any of embodiment 1 to 4, wherein the gaseous substrate supply conduit further comprises i) a gaseous substrate sensor to monitor a gaseous substrate concentration and transmits the concentration to the control system, ii) a gaseous substrate shut-off actuated valve in communication with the control system, and iii) a gaseous substrate flow' control valve in communication with the control system.

[0138] Embodiment 6. The apparatus of any of embodiment 1 to 5, wherein the gaseous substrate supply unit further comprises two supply units; a first gaseous substrate supply unit which is in communication with a first gaseous substrate supply conduit and is in communication with the at least one gaseous substrate inlet on the bioreactor vessel ; and a second gaseous substrate supply unit supplying a second gaseous substrate a a gas comprising CO2 which is in communication with a second gaseous substrate supply conduit in communication with a second gaseous substrate inlet on the bioreactor vessel located on the exterior casing and below the surface of the liquid nutrient medium.

[0139] Embodiment 7. The apparatus of any of embodiment 1 to 6, wherein the bioreactor further comprises one or more gaseous substrate sensors for measuring a concentration of the gaseous substrate in the culture, and in communication with a flow controller located on the gaseous substrate supply conduit.

[0140] Embodiment 8. The apparatus of any of embodiment 1 to 7, wherein the bioreactor further comprises one or more gaseous substrate sensors for measuring a concentration of the first and second gaseous substrate in the culture, and in communication with a flow controller located on the first and second gaseous substrate conduits.

[0141] Embodiment 9. The apparatus of any of embodiment 1 to 8, wherein the at least one vent gas sample conduit further 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 vent gas sample conduit measure concentration of CH4 in the gas headspace and are in communication with the control system.

[0142] Embodiment 10. The apparatus of any of embodiment 1 to 9, wherein the control system continuously receives a measured concentration of O2 in the gas headspace from the at least one oxygen sensor, located on the vent gas sample outlet conduit or in the headspace, through an O2 sensor transmitter located on the vent gas sample outlet conduit or in the headspace; the control system comprising 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 by sending a signal to the gas comprising O2 flow control valve or the gas comprising O2 shut off valve.

[0143] Embodiment 11. The apparatus of any of embodiment 1 to 10, further comprising at least one purge gas inlet located above the top surface of the liquid nutrient medium in the gas headspace and in fluid communication with at least one purge gas conduit, the purge gas conduit in fluid communication with a purge gas supply unit, the purge gas supply conduit further comprising a purge gas shut-off valve, wherein the purge gas supply unit supplies an inert gas selected from N2, Ar, CO2, or any combination thereof.

[0144] Embodiment 12. The apparatus of any of embodiment 1 to 11, wherein the control system controls the flow of purge gas from the purge gas supply unit to the bioreactor and the flow of the gas comprising O2 from the gas comprising O2 supply unit to the bioreactor the control system comprising a computer processor which executes a computer program implemented process for controlling the flow rate of the purge gas and the gas comprising O2 based on the measured concentration of oxygen in the gas headspace.

[0145] Embodiment 13. The apparatus of any of embodiment 1 to 12, further comprising at least one diluent inlet located on the exterior casing and 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, wherein the diluent supply unit supplies a diluent selected from a gas selected from H2, CO2 N2, Ar, or any combination thereof.

[0146] Embodiment 14. The apparatus of any of embodiment 1 to 13. wherein the control system controls the flow of diluent from the diluent supply unit to the bioreactor and the flow of the gas comprising O2 from the gas comprising O2 supply unit to the bioreactor, the control system further comprising a computer processor which executes a computer program implemented process for controlling the flow rate of the diluent and the gas comprising O2 based on the measured concentration of oxygen in the gas headspace.

[0147] Embodiment 15. A method for aerobic gas fermentation comprising introducing at least two gaseous feed streams via at least two gaseous feed stream conduits to a bioreactor comprising a liquid nutrient medium comprising a culture of a microorganism, wherein at least one gaseous feed stream comprises a gas comprising O2, and at least one gaseous feed stream comprises a gaseous substrate selected from a gas comprising H2, CO2, CEU or any combination thereof, and fermenting the gaseous substrate plus the gas comprising O2 to produce microbial biomass and / or at least one chemical product and a tail gas which forms a headspace above the liquid nutrient medium in the bioreactor; measuring gaseous oxygen concentration in the headspace of the bioreactor; transmitting the oxygen concentration to a control system which determines whether the measured concentration is above a first set point or determines a rate of change of O2 concentration is above a predetermined value and sends a signal to an O2 flow control valve located on the gas comprising O2 conduit to reduce the flow of the gas comprising O2 feed stream.

[0148] Embodiment 16. The method of embodiment 15, wherein the gaseous substrate is a gas comprising H2 and the first set point is from about 1 vol % O2 to about 2 vol % O2.

[0149] Embodiment 17. The method of embodiment 15 or 16, wherein the gaseous substrate comprises a gas comprising H2 and a gas comprising CO2 and both gases are combined into one gaseous feed stream which is fed into the bioreactor or the gas comprising H2 and the gas comprising CO2 are fed as separate gaseous feed streams into the bioreactor.

[0150] Embodiment 18. The method of any of embodiments 15 to 17, wherein the gas comprising H2 has a concentration from about 1 vol % to 100 vol %. the gas comprising CO2has a concentration of CO2 from about 1 vol % to about 100 vol %, and the gas comprising O2 has a concentration of O2 from about 20 vol % to about 100 vol %.

[0151] Embodiment 19. The method of any of embodiments 15 to 18, further comprising after the flow of the gas comprising O2 feed stream is reduced, waiting a predetermined time and determining if the O2 concentration in the headspace has dropped below the first set point.

[0152] Embodiment 20. The method of any of embodiments 15 to 19, wherein the predetermined time varies from about 30 seconds to about 10 minutes.

[0153] Embodiment 21. The method of any of embodiments 15 to 20, further comprising measuring the concentration of the at least one gaseous substrate in the feed stream and if the concentration is below a set point shutting off or reducing the flow of the gas comprising O2 feed stream into the bioreactor.

[0154] Embodiment 22. The method of any of embodiments 15 to 21, further comprising measuring a flow in a vent gas sample conduit and if low flow or no flow is detected initiating an interlock action whereby the flow of the gas comprising O2 feed stream and the flow of the gaseous feed stream comprising the gaseous substrate into the bioreactor is reduced if low flow is detected or shut off if no flow is detected.

[0155] Embodiment 23. The method of any of embodiments 15 to 22, further comprising measuring O2 concentration of the gas comprising O2 in the at least one gaseous feed stream, and if the O2 concentration is higher than a predetermined value or rate of change of concentration of O2 is above a predetermined value initiating an interlock action whereby the flow of the gas comprising O2 into the bioreactor is either reduced or shut off and the flow of the gaseous substrate into the bioreactor is reduced or shut off.

[0156] Embodiment 24. The method of any of embodiments 15 to 22, further comprising measuring the gaseous substrate concentration in the at least one gaseous feed stream, and when the measured gaseous substrate concentration is below a predetermined value initiating an interlock action whereby the flow of the gas comprising O2 into the bioreactor vessel is reduced or shut off.

[0157] Embodiment 25. The method of any of embodiments 15 to 24, wherein the gaseous substrate further comprises CO.

[0158] Embodiment 26. The method of any of embodiments 15 to 25, further comprising determining if the oxygen concentration in the headspace is above a second set point, the second set point having a higher value than the first set point, initiating an interlock action whereby the flow of the gas comprising O2 feed stream and the gaseous substrate feed stream into the bioreactor is shut off.

[0159] Embodiment 27. The method of any of embodiments 15 to 26, wherein the gaseous substrate is a gas comprising H2 and the second set point is greater than 1 vol % O2 to about 2.75 vol % O2.

[0160] Embodiment 28. The method of any of embodiments 15 to 27, wherein the gaseous substrate is a gas comprising CH4 and the first set point is in the range from about 5 vol % O2 to about 15 vol % O2 .

[0161] Embodiment 29. The method of any of embodiments 15 to 28, wherein the gaseous substrate is a gas comprising CH4 and the second set point is in the range from about 6 vol % O2 to about 18 vol % O2.

[0162] Embodiment 30. The method of any of embodiments 15 to 29, further comprising flowing a purge gas into the headspace of the bioreactor when the second set point is above 2.5 vol % O2 and wherein the purge gas is selected from Ar, N2, CO2 or any combination thereof.

[0163] Embodiment 31. The method of any of embodiments 15 to 30, further comprising introducing a diluent gas stream into the gas headspace to dilute the concentration of O2 in the gas headspace, wherein the diluent gas stream comprises a gas selected from H2, CO2 N2, Ar, or any combination thereof.

[0164] Embodiment 32. The method of any of embodiments 15 to 31, further comprising measuring a concentration of the gaseous substrate in the liquid nutrient medium and adjusting the gaseous substrate flow rate into the bioreactor based on the concentration of the gaseous substrate.

Claims

What is claimed:

1. An apparatus for aerobic gas fermentation, 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 gas 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;- a vent gas sample outlet located approximate to the top of the bioreactor vessel and within the gas headspace, and in fluid communication with at least one vent gas sample conduit comprising at least one oxygen sensor and optionally at least one oxygen sensor located within the gas headspace, the at least one oxygen sensor in the gas headspace or on the vent gas sample conduit measure a concentration of oxygen in the gas 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.

2. The apparatus of claim 1, wherein the at least one vent gas sample conduit further comprises at least one vent gas sample flow monitors and / or transmitters for monitoring the flow of the vent gas in the vent gas sample conduit.

3. The apparatus of claim 1. wherein the gas comprising O2 supply conduit further comprises:a) an O2 gas sensor to monitor the O2 concentration and transmit the O2 concentration to the control system; b) a gas comprising O2 shut-off actuated valve in communication with the control system; and c) a gas comprising O2 flow control valve in communication with the control system.

4. The apparatus of claim 1, wherein the gaseous substrate supply conduit further comprises i) a gaseous substrate sensor to monitor a gaseous substrate concentration and transmit the concentration to the control system, ii) a gaseous substrate shut-off actuated valve in communication with the control system, and iii) a gaseous substrate flow control valve in communication with the control system and / or wherein the gaseous substrate supply unit further comprises two supply units; a first gaseous substrate supply unit which is in communication with a first gaseous substrate supply conduit and is in communication with the at least one gaseous substrate inlet on the bioreactor vessel ; and a second gaseous substrate supply unit supplying a second gaseous substrate comprising CO2 which is in communication with a second gaseous substrate supply conduit in communication with a second gaseous substrate inlet on the bioreactor vessel located on the exterior casing and below the surface of the liquid nutrient medium.

5. The apparatus of claim 1. wherein the bioreactor further comprises one or more gaseous substrate sensors for measuring a concentration of the gaseous substrate in the culture, and in communication with a flow controller located on the gaseous substrate supply conduit.

6. The apparatus of claim 4, wherein the bioreactor further comprises one or more gaseous substrate sensors for measuring a concentration of the first and second gaseous substrate in the culture, and in communication with a flow controller located on the first and second gaseous substrate conduits.

7. The apparatus of claim 1, wherein the at least one vent gas sample conduit further comprises at least one CT sensor and optionally at least one CH4 sensor located within the gas headspace, the CH4 sensor in the gas headspace or on the vent gas sample conduit measure concentration of CH4 in the gas headspace and are in communication with the control system.

8. The apparatus of claim 1, wherein the control system continuously receives a measured concentration of O2 in the gas headspace from the at least one oxygensensor, located on the vent gas sample outlet conduit or in the headspace, through an O2 sensor transmitter located on the vent gas sample outlet conduit or in the headspace; the control system comprising 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 by sending a signal to the gas comprising O2 flow control valve or the gas comprising O2 shut off valve.

9. The apparatus of claim 1, further comprising at least one purge gas inlet located above the top surface of the liquid nutrient medium in the gas headspace and in fluid communication with at least one purge gas conduit, the purge gas conduit in fluid communication with a purge gas supply unit, the purge gas supply conduit further comprising a purge gas shut-off valve, wherein the purge gas supply unit supplies an inert gas selected from N2, Ar, CO2, or any combination thereof and / or wherein the control system controls the flow of purge gas from the purge gas supply unit to the bioreactor and the flow of the gas comprising O2 from the gas comprising O2 supply unit to the bioreactor, the control system comprising a computer processor which executes a computer program implemented process for controlling the flow rate of the purge gas and the gas comprising O2 based on the measured concentration of oxygen in the gas headspace and / or further comprising at least one diluent inlet located on the exterior casing and 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, wherein the diluent supply unit supplies a diluent selected from a gas selected from N2, Ar, CO2. or any combination thereof and / or wherein the control system controls the flow of diluent from the diluent supply unit to the bioreactor and the flow of the gas comprising O2 from the gas comprising O2 supply unit to the bioreactor, the control system further comprising a computer processor which executes a computer program implemented process for controlling the flow rate of the diluent and the gas comprising O2 based on the measured concentration of oxygen in the gas headspace.

10. A method for aerobic gas fermentation comprising: introducing at least two gaseous feed streams via at least two gaseous feed stream conduits to a bioreactor comprising a liquid nutrient medium comprising a culture of a microorganism, wherein at least one gaseous feed streamcomprises a gas comprising O2, and at least one gaseous feed stream comprises a gaseous substrate selected from a gas comprising H2, CO2, CH4 or any combination thereof and fermenting the gaseous substrate plus the gas comprising O2, to produce microbial biomass and / or at least one chemical product and a tail gas which forms a headspace above the liquid nutrient medium in the bioreactor;- measuring gaseous oxygen concentration in the headspace of the bioreactor; transmitting the oxygen concentration to a control system which determines whether the measured concentration is above a first set point or determines a rate of change of O2 concentration is above a predetermined value and sends a signal to an O2 flow control valve located on the gas comprising O2 conduit to reduce the flow of the gas comprising O2 feed stream.

11. The method of claim 10, wherein the gaseous substrate is a gas comprising H2 and the first set point is from about 1 vol % O2 to about 2 vol % O2.

12. The method of claim 10. wherein the gaseous substrate comprises agas comprising H2 and a gas comprising CO2 and both gases are combined into one gaseous feed stream which is fed into the bioreactor or the gas comprising H2 and the gas comprising CChare fed as separate gaseous feed streams into the bioreactor and / or wherein the gas comprising H2 has a concentration from about 1 vol % to 100 vol %. the gas comprising CO2 has a concentration of CO2 from about 1 vol % to about 100 vol %, and the gas comprising O2 has a concentration of O2 from about 20 vol % to about 100 vol %.

13. The method of claim 10, further comprising after the flow of the gas comprising O2 feed stream is reduced, waiting a predetermined time and determining if the O2 concentration in the headspace has dropped below the first set point and wherein the predetermined time varies from about 30 seconds to about 10 minutes.

14. The method of claim 10, further comprising measuring a flow' in a vent gas sample conduit and if low flow or no flow is detected initiating an interlock action whereby the flow of the gas comprising O2 feed stream and the flow of the gaseous feed stream comprising the gaseous substrate into the bioreactor is reduced if low- flow is detected or shut off if no flow is detected.

15. The method of claim 10, further comprising measuring O2 concentration of the gas comprising O2 in the at least one gaseous feed stream, and if the O2 concentration is higher than a predetermined value or rate of change ofconcentration of O2 is above a predetermined value initiating an interlock action whereby the flow of the gas comprising O2 into the bioreactor is either reduced or shut off and the flow of the gaseous substrate into the bioreactor is reduced or shut off and / or measuring the gaseous substrate concentration in the at least one gaseous feed stream, and when the measured gaseous substrate concentration is below a predetermined value initiating an interlock action whereby the flow of the gas comprising O2 into the bioreactor vessel is reduced or shut off and / or further comprising measuring a flow in a gaseous feed stream conduit and if low flow or no flow is detected initiating an interlock action whereby the flow of the gas comprising O2 feed stream and the flow of the gaseous feed stream comprising the gaseous substrate into the bioreactor is reduced if low flow is detected or shut off if no flow is detected.

16. The method of claim 10, further comprising determining if the oxygen concentration in the headspace is above a second set point, the second set point having a higher value than the first set point, initiating an interlock action whereby the flow of the gas comprising O2 feed stream and the gaseous substrate feed stream into the bioreactor is shut off and wherein when the gaseous substrate is a gas comprising H2, the second set point is greater than 1 vol % O2 to about 2.75 vol % O2.

17. The method of claim 10, wherein the gaseous substrate is a gas comprising CH4 and the first set point is in the range from about 5 vol % O2 to about 15 vol % O2 and / or wherein when the gaseous substrate is a gas comprising CH4 and a second set point is in the range from about 6 vol % O2 to about 18 vol % O2.

18. The method of claim 17. further comprising flowing a purge gas into the headspace of the bioreactor when the second set point is above 2.5 vol % O2 and wherein the purge gas is selected from Ar, N2, CO2 or any combination thereof.

19. The method of claim 10, further comprising introducing a diluent gas stream into the gas headspace to dilute the concentration of O2 in the gas headspace, wherein the diluent gas stream comprises a gas selected from H2, CO2 N2, Ar, or any combination thereof.

20. The method of claim 10, further comprising measuring a concentration of the gaseous substrate in the liquid nutrient medium and adjusting the gaseous substrate flow rate into the bioreactor based on the concentration of the gaseous substrate.

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