PROCESS FOR PRODUCING ONE OR MORE FERMENTATION PRODUCTS

Abstract

The present invention relates to a process for producing one or more fermentation products in a multi-stage process, including an inoculation reactor and at least one bioreactor. The inoculation reactor is fed with a gaseous substrate containing C1 with a reduced amount of hydrogen. The hydrogen is reduced to increase the proportion of CO in the C1-containing gaseous substrate that is fed to the inoculation reactor. The inoculation reactor ferments the CO-rich C1-containing gaseous substrate and produces an inoculum, which is fed to at least one bioreactor. The bioreactor receives the C1-containing gaseous substrate, which may or may not contain reduced amounts of hydrogen, to produce one or more fermentation products. By feeding a CO-rich C1-containing gaseous substrate to the inoculation reactor, both the inoculation reactor and the subsequent bioreactor (or bioreactors) have the capacity to have greater product stability and selectivity.

Description

36 PROCESS FOR PRODUCING ONE OR MORE FERMENTATION PRODUCTS CROSS-REFERENCE TO A RELATED REQUEST

[001] The application claims the benefit of Provisional Application No. US 62 / 556.099, filed on September 8, 2017, the contents of which are incorporated herein by reference. FIELD OF THE INVENTION

[002] The present invention relates to a process for producing one or more fermentation products via a multi-stage gas fermentation process, including an inoculation reactor and at least one bioreactor. In particular, the invention relates to a process whereby a gaseous substrate containing CO-rich C1 is supplied to the inoculation reactor for the production of an inoculum. BACKGROUND OF THE INVENTION

[003] Carbon dioxide (CO2) is responsible for approximately 76% of global greenhouse gas emissions from human activities, with methane (16%), nitrous oxide (6%), and fluorinated gases (2%) accounting for the balance (United States Environmental Protection Agency). Reducing greenhouse gas emissions, primarily CO2, is crucial to halting the progression of global warming and the accompanying climate and weather changes.

[004] It has long been recognized that catalytic processes, such as the Fischer-Tropsch process, can be used to convert gases containing carbon dioxide (CO2), carbon monoxide (CO), and / or hydrogen (H2), such as industrial waste gas or synthesis gas, into a variety of fuels and chemicals. Recently, however, gas fermentation has emerged as an alternative platform for the biological fixation of these gases. In particular, C1 fixation microorganisms have been shown to convert gases containing CO2, CO, and / or H2 into products, Petition 870200030711, dated 06 / 03 / 2020, page 12 / 61 / 36 such as ethanol and 2,3-butanediol.

[005] Such gases may be derived, for example, from industrial processes, including carbohydrate fermentation gas, cement manufacturing gas, paper and pulp manufacturing, steelmaking, petroleum refining and associated processes, petrochemical production, coke production, anaerobic or aerobic digestion, synthesis gas (derived from sources including, but not limited to, biomass, liquid waste streams, solid waste streams, municipal streams, fossil resources including natural gas, coal and oil), natural gas extraction, oil extraction, metallurgical processes, for the production and / or refining of aluminum, copper and / or ferroalloys, geological reservoirs and catalytic processes (derived from steam sources including, but not limited to, steam methane reforming, steam naphtha reforming, petroleum coke gasification, catalyst regeneration - fluid catalyst cracking, catalyst regeneration - naphtha reforming and dry methane reforming).

[006] With specific industrial processes, the gas composition may not be ideal for fermentation. When the gas composition is not ideal, cell growth, product selectivity, and stability may be less than ideal.

[007] Therefore, there remains a need for an invention that optimizes the gas composition of industrial processes to promote cell growth, product selectivity and stability in a downstream fermentation process. BRIEF SUMMARY OF THE INVENTION

[008] The invention provides a process for producing one or more fermentation products, wherein a gaseous substrate containing CO₂-rich C₁ is passed to an inoculation reactor comprising a liquid nutrient medium containing a culture of one or more C₁-fixing microorganisms in which the gaseous substrate containing CO₂-rich C₁ is Petition 870200030711, dated 06 / 03 / 2020, page 13 / 61 / 36 fermented to produce an inoculum, at least a portion of the inoculum is passed to a bioreactor system, wherein the bioreactor system defines at least one bioreactor containing a culture of one or more C1-fixing microorganisms in a liquid nutrient medium, a gaseous substrate containing H2-rich C1 is passed to the bioreactor system, wherein the gaseous substrate containing H2-rich C1 is fermented to produce at least one fermentation product.

[009] In particular embodiments, the gaseous substrate containing C1 rich in CO to be passed to the inoculation reactor comprises H2 in a molar ratio of H2:CO of less than 1:1.

[0010] In certain cases, the gaseous substrate containing CO-rich C1 to be passed to the inoculation reactor comprises H2 at an H2:CO molar ratio of less than 0.5:1.

[0011] Preferably, the gaseous substrate containing CO-rich C1 to be passed to the inoculation reactor comprises H2 at an H2:CO molar ratio between 0.02:1 and 1:1. In certain embodiments, the H2:CO molar ratio is between 0.05:1 and 1:1, or 0.15:1 and 1:1, or 0.25:1 and 1:1, or 0.35:1 and 1:1, or 0.45:1 and 1:1, or 0.55:1 and 1:1, or 0.65:1 and 1:1, or 0.75:1 and 1:1, or 0.85:1 and 1:1, or 0.95:1 and 1:1.

[0012] In particular embodiments, the gaseous substrate containing H2-rich C1 to be passed to the bioreactor system comprises H2 in an H2:CO molar ratio of at least 1.1:1.

[0013] Preferably, the gaseous substrate containing H2-rich C1 to be passed to the bioreactor system comprises H2 in an H2:CO molar ratio between 1.1:1 and 6:1. In certain embodiments, the H2:CO molar ratio is between 1.5:1 and 6:1, or 2:1 and 6:1, or 2.5:1 and 6:1, or 3:1 and 6:1, or 3.5:1 and 6:1, or 4:1 and 6:1, or 4.5:1 and 6:1, or 5:1 and 6:1.

[0014] In at least one embodiment, the C1-fixing microorganism in the inoculation reactor or bioreactor system, or both, is Petition 870200030711, dated 06 / 03 / 2020, p. 14 / 61 / 36 a carboxidotrophic bacterium.

[0015] In embodiments where the C1-fixing microorganism is carboxidotrophic, the carboxidotrophic bacterium may be selected from the group consisting of Moorella, Clostridium, Ruminococcus, Acetobacterium, Eubacterium, Butyribacterium, Oxobacter, Methanosarcina and Desulfotomaculum.

[0016] Preferably, the carboxidotrophic bacterium is Clostridium autoethanogenum.

[0017] In at least one embodiment, the bioreactor system comprises one or more primary bioreactors connected to one or more secondary bioreactors.

[0018] Preferably, the process provides for the passage of at least a portion of a gaseous substrate containing C1 to an inoculation reactor and at least a portion of the gaseous substrate containing C1 to a bioreactor, wherein the gaseous substrate containing C1 in the inoculation reactor is fermented to produce an inoculum, wherein at least a portion of the inoculum is passed to at least one bioreactor, wherein the gaseous substrate containing C1 in the bioreactor is fermented to produce at least one fermentation product, and wherein the gaseous substrate containing C1 that is passed to the inoculation reactor is subjected to at least one H2 removal process before being passed to the inoculation reactor.

[0019] In particular embodiments, the gaseous substrate containing C1 to be passed to the inoculation reactor comprises H2 in a molar ratio of H2:CO of less than 1:1.

[0020] In certain cases, the gaseous substrate containing C1 that is passed to the inoculation reactor comprises H2 in a molar ratio of H2:CO of less than 0.8:1.

[0021] In certain cases, the gaseous substrate containing C1 to be passed Petition 870200030711, dated 06 / 03 / 2020, page 15 / 61 / 36 for the inoculation reactor comprising H2 in a molar ratio of H2:CO of less than 0.5:1.

[0022] Preferably, the gaseous substrate containing C1 that is passed to the inoculation reactor comprises H2 in a molar ratio of H2:CO between 0.02:1 and 1:1. In certain embodiments, the molar ratio of H2:CO is between 0.05:1 and 1:1, or 0.15:1 and 1:1, or 0.25:1 and 1:1, or 0.35:1 and 1:1, or 0.45:1 and 1:1, or 0.55:1 and 1:1, or 0.65:1 and 1:1, or 0.75:1 and 1:1, or 0.85:1 and 1:1, or 0.95:1 and 1:1.

[0023] In certain embodiments, the H2 removal process comprises at least one pressure-swinging adsorption process.

[0024] In certain embodiments, the H2 removal process comprises at least one membrane separation module.

[0025] Preferably, at least a portion of the gaseous substrate containing C1 is derived from an industrial source.

[0026] In certain cases, at least a portion of the gaseous substrate containing C1 may be derived from at least one industrial source selected from the group consisting of carbohydrate fermentation, gas fermentation, cement manufacturing, paper and pulp manufacturing, steelmaking, petroleum refining and associated processes, petrochemical production, coke production, anaerobic or aerobic digestion, synthesis gas, natural gas extraction, oil extraction, metallurgical processes for the production and / or refining of aluminum, copper and / or ferroalloys, geological reservoirs and catalytic processes.

[0027] Preferably, the process produces at least one fermentation product selected from the group consisting of: ethanol, acetate, butanol, butyrate, 2,3-butanediol, 1,3-butanediol, lactate, butene, butadiene, methyl ethyl ketone, ethylene, acetone, isopropanol, lipids, 3-hydroxypropionate, isoprene, fatty acids, 2-butanol, 1,2-propanediol, 1-propanol, monoethylene glycol, isobutene and C6-C14 alcohols. Petition 870200030711, dated 06 / 03 / 2020, page 16 / 61 / 36

[0028] In at least one embodiment, one or more fermentation products are further converted into at least one component of diesel fuel, aviation fuel, gasoline, propylene, nylon 66, rubber and / or resins.

[0029] In particular embodiments, at least one fermentation product is microbial biomass. In certain cases, this microbial biomass may be further processed to produce at least one component of animal feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a schematic flowchart depicting the integration of a hydrogen removal process, an inoculation reactor, and a bioreactor system.

[0031] Figure 2 is a schematic flowchart depicting the integration of a hydrogen removal process, an inoculation reactor, and a bioreactor system, wherein the hydrogen removal process is upstream of both the inoculation reactor and the bioreactor system, according to one aspect of the invention.

[0032] Figure 3 is a schematic flowchart that further depicts two water-gas exchange processes and an adsorption process with pressure swing upstream of the bioreactor system, in which a water-gas exchange process is bypassed, according to one aspect of the invention.

[0033] Figure 4 is a schematic flowchart that further depicts two water-gas exchange processes and an adsorption process with pressure swing upstream of the bioreactor system, according to one aspect of the invention.

[0034] Figure 5 is a schematic flowchart that further depicts additional hydrogen removal processes upstream of the inoculation reactor, according to one aspect of the invention. Petition 870200030711, dated 06 / 03 / 2020, page 17 / 61 / 36

[0035] Figures 6a and 6b are graphs showing metabolite production and gas uptake in a first bioreactor according to Example 1.

[0036] Figures 7a and 7b are graphs showing metabolite production and gas uptake in a second bioreactor according to Example 1.

[0037] Figures 8a and 8b are graphs showing metabolite production and gas uptake in a first bioreactor according to Example 2.

[0038] Figures 9a and 9b are graphs showing metabolite production and gas uptake in a second bioreactor according to Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0039] The inventors have identified that by optimizing the composition of a gas stream supplied to the inoculation reactor, cell growth, product selectivity, and stability are optimized in both the inoculation reactor and the subsequent bioreactor system. In particular, the inventors found optimal cell growth, product selectivity, and stability when the gas stream supplied to the inoculation reactor comprises a reduced amount of hydrogen. DEFINITIONS

[0040] Unless defined otherwise, the following terms, used throughout this descriptive report, are defined as follows:

[0041] C1 refers to a one-carbon molecule, for example, CO, CO2, CH4, or CH3OH. C1-oxygenated refers to a one-carbon molecule that also comprises at least one oxygen atom, for example, CO, CO2, or CH3OH. C1-carbon source refers to a carbon molecule that serves as a partial or sole source of carbon for the Petition 870200030711, dated 06 / 03 / 2020, p. 18 / 61 / 36 microorganism of the invention. For example, a carbon source C1 may comprise one or more of CO, CO2, CH4, CH3OH or CH2O2. Preferably, the carbon source C1 comprises one or both of CO or CO2. A “C1-fixing microorganism” is a microorganism capable of producing one or more products from a carbon source C1. Typically, the microorganism of the invention is a C1-fixing microorganism.

[0042] Gaseous substrates containing C1 include any gas exiting an industrial process that comprises C1. In many cases, the gaseous substrate containing C1 comprises CO, H2, CO2, or combinations thereof. The gaseous substrate will typically contain a significant proportion of CO, preferably at least about 5% to about 100% CO by volume. The gaseous substrate may contain a significant proportion of hydrogen. For example, in particular embodiments, the substrate may comprise a ratio of approximately 2:1, or 1:1, or 1:2 of H2:CO. In one embodiment, the substrate comprises about less than 30% or less by volume of H2, 20% or less of H2 by volume, about 15% or less by volume of H2, or about 10% or less by volume of H2. The substrate may also contain some CO2, for example, from about 1% to about 80% by volume of CO2, or from 1% to about 30% by volume of CO2. In one embodiment, the substrate comprises less than or equal to 20% by volume of CO2.In particular embodiments, the substrate comprises less than or equal to about 15% by volume of CO2, less than or equal to about 10% by volume of CO2, less than or equal to about 5% by volume of CO2, or substantially no CO2. Furthermore, the gaseous substrate containing C1 may contain one or more of oxygen (O2), nitrogen (N2), and / or methane (CH4).

[0043] Although the substrate is typically gaseous, the substrate can also be provided in alternative forms. For example, the substrate Petition 870200030711, dated 06 / 03 / 2020, page 19 / 61 / 36, can be dissolved in a liquid saturated with a gas containing CO using a microbubble dispersion generator. As a further example, the substrate can be adsorbed onto a solid support.

[0044] The term “cossubstrate” refers to a substance that, while not necessarily the primary energy and relevant source for product synthesis, can be used for product synthesis when added to another substrate, such as the primary substrate.

[0045] The substrate and / or carbon source C1 may be a waste gas obtained as a byproduct of an industrial process or from some other source, such as automobile exhaust gases or biomass gasification. In certain embodiments, the industrial process is selected from the group consisting of emissions from carbohydrate fermentation gases, gas fermentation, emissions from cement manufacturing, paper and pulp manufacturing, steel production, petroleum refining and associated processes, petrochemical production, coke production, anaerobic or aerobic digestion, synthesis gas (derived from sources including, but not limited to, biomass, liquid waste streams, solid waste streams, municipal streams, fossil resources including natural gas, coal and oil), natural gas extraction, oil extraction, metallurgical processes, for the production and / or refining of aluminum, copper and / or ferroalloys,Geological reservoirs and catalytic processes (derived from steam sources, including, but not limited to, steam methane reforming, steam naphtha reforming, petroleum coke gasification, catalyst regeneration - fluid catalyst cracking, catalyst regeneration - naphtha reforming and dry methane reforming). In these embodiments, the substrate and / or carbon source C1 can be captured from the industrial process before being emitted into the atmosphere, using any convenient method.

[0046] “Gas stream refers to any substrate stream Petition 870200030711, dated 06 / 03 / 2020, page 20 / 61 / 36 with the capacity to be transmitted, for example, from one module to another, from a module to a bioreactor, from a module to an inoculation reactor, from one process to another process, and / or from a module to a carbon capture medium.

[0047] The term carbon capture, as used in this document, refers to the sequestration of carbon compounds, including CO2 and / or CO, from a stream comprising CO2 and / or CO and comprising: convert CO2 and / or CO into products; or convert CO2 and / or CO into substances suitable for long-term storage; or trap CO2 and / or CO in substances suitable for long-term storage; or a combination of these processes.

[0048] Reagents, as used in this document, refers to a substance that is part of a chemical reaction and undergoes changes during the reaction. In particular embodiments, reactants include, but are not limited to, CO and / or H2.

[0049] Hydrogen removal and similar processes include technologies capable of removing and / or separating hydrogen from the gaseous substrate containing C1. In particular embodiments, a pressure swing adsorption process and / or a membrane separation process are used as the hydrogen removal process.

[0050] The term “bioreactor”, “bioreactor system” and the like include a fermentation device consisting of one or more vessels and / or towers or piping arrangements, which include the Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Spray Reactor (TBR), Bubble Column, Gas Lift Fermenter, Static Mixer, a closed-loop circulated reactor, a membrane reactor, such as a Hollow Fiber Membrane Bioreactor (HFM BR) or other vessel or Petition 870200030711, dated 06 / 03 / 2020, page 21 / 61 / 36 another suitable device for gas-liquid contact. The bioreactor is preferably adapted to receive a gaseous substrate comprising CO or CO2 or H2 or mixtures thereof. The bioreactor may comprise several reactors (stages), in parallel or in series. Preferably, the bioreactor is configured to receive an inoculum from an incubation reactor. Preferably, the bioreactor is configured as a production reactor, in which most of the fermentation products are produced.

[0051] The term inoculation reactor, inoculator, seed reactor and the like includes a fermentation device for establishing and promoting cell growth. The inoculation reactor is preferably adapted to receive a gaseous substrate comprising CO or CO2 or H2 or mixtures thereof. Preferably, the inoculation reactor is a reactor in which cell growth is initiated for the first time. In various embodiments, the inoculation reactor is the location where the growth cells are previously recovered. In various embodiments, the inoculator initiates cell growth of one or more microorganisms to produce an inoculum, which can be transferred to the bioreactor system in which each bioreactor is configured to promote the production of one or more fermentation products. In certain cases, the inoculator has a reduced volume when compared to one or more subsequent bioreactors.

[0052] “Nutrient media or nutrient medium is used to describe the bacterial growth medium. Generally, this term refers to a medium that contains nutrients and other components appropriate for the growth of a microbial culture. The term “nutritive” includes any substance that can be used in a metabolic pathway of a microorganism. Exemplary nutrients include potassium, B vitamins, trace metals, and amino acids.

[0053] The term fermentation broth or broth is intended to encompass the mixture of components, including nutrient medium and a culture or Petition 870200030711, dated 06 / 03 / 2020, p. 22 / 61 / 36 one or more microorganisms. It should be noted that the term microorganism and the term bacteria are used interchangeably throughout the document.

[0054] The term inoculum is intended to encompass the fermentation broth initially cultivated in the inoculation reactor which is then passed on to one or more subsequent bioreactors to seed the one or more subsequent bioreactors. Preferably, the inoculum is used by the one or more bioreactors to produce one or more fermentation products.

[0055] The term desired composition is used to refer to the level and types of components desired in a substance, such as, for example, a gas stream. More particularly, a gas is considered to have a “desired composition” if it contains a certain component (e.g., CO, H2, and / or CO2) and / or contains a certain component in a specific proportion and / or does not contain a specific component (e.g., a constituent harmful to microorganisms) and / or does not contain a specific component in a specific proportion. More than one component may be considered when determining the possibility of a gas stream having the desired composition. In one or more embodiments, the “desired composition” of the gaseous substrate containing C1 is defined in terms of a molar ratio of H2:CO.In several embodiments, the desired composition of the gaseous substrate containing C1 that is passed to the inoculation reactor differs from the desired composition of the gaseous substrate containing C1 that is passed to the bioreactor system.

[0056] The terms “increased efficiency”, “greater efficiency” and the like, when used in relation to a fermentation process, include, but are not limited to, increasing one or more growth rates of microorganisms that catalyze fermentation, the growth and / or production rate of product at high product concentrations, the volume of product Petition 870200030711, dated 06 / 03 / 2020, page 23 / 61 / 36 desired product produced by volume of substrate consumed, the production rate or level of production of the desired product and the relative proportion of the desired product produced compared to other fermentation by-products.

[0057] Unless the context requires otherwise, the phrases fermentation, fermentation process or fermentation reaction and the like, as used herein, are intended to encompass both the growth phase and the biosynthesis phase of the gaseous substrate product.

[0058] A “microorganism” is a microscopic organism, especially a bacterium, Archaea, virus or fungus. The microorganism of the invention is typically a bacterium. As used herein, the term “microorganism” shall be interpreted to encompass bacteria.

[0059] A “parental microorganism” is a microorganism used to generate a microorganism of the invention. The parental microorganism may be a naturally occurring microorganism (e.g., a wild-type microorganism) or a microorganism that has been previously modified (e.g., a mutant or recombinant microorganism). The microorganism of the invention may be modified to express or overexpress one or more enzymes that were not expressed or overexpressed in the parental microorganism. Similarly, the microorganism of the invention may be modified to contain one or more genes that were not contained by the parental microorganism. The microorganism of the invention may also be modified to not express or to express lower amounts of one or more enzymes that were expressed in the parental microorganism. In one embodiment, the parental microorganism is Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei.In a preferred embodiment, the parent microorganism is Clostridium autoethanogenum LZ1561, which was deposited on June 7th. Petition 870200030711, dated 06 / 03 / 2020, page 24 / 61 / 36 In 2010, with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) located at InlioffenstraB 7B, D-38124 Braunschweig, Germany on June 7, 2010, under the terms of the Budapest Treaty and which received accession number DSM23693. This strain is described in International Patent Application No. PCT / NZ2011 / 000144, published as document WO 2012 / 015317.

[0060] The term derived from indicates that a nucleic acid, protein, or microorganism is modified or adapted from a different nucleic acid, protein, or microorganism (for example, a parenteral or wild-type microorganism) in order to produce a new nucleic acid, protein, or microorganism. Such modifications or adaptations typically include insertion, deletion, mutation, or substitution of nucleic acids or genes. Generally, the microorganism of the invention is derived from a parent microorganism. In one embodiment, the microorganism of the present invention is derived from Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferred embodiment, the microorganism of the invention is derived from Clostridium autoethanogenum LZ1561, which is deposited under accession number DSMZ DSM23693.

[0061] Wood-Ljungdahl refers to the Wood-Ljungdahl carbon fixation pathway as described, i.e., by Ragsdale, Biochim Biophys Acta, 1784:1,873 to 1,898, 2008. Wood-Ljungdahl microorganisms predictably refer to microorganisms that contain the Wood-Ljungdahl pathway. Generally, the microorganism of the invention contains a native Wood-Ljungdahl pathway. In the present document, a Wood-Ljungdahl pathway may be an unmodified, native Wood-Ljungdahl pathway or it may be a Wood-Ljungdahl pathway with some degree of genetic modification (e.g., overexpression, heterologous expression, knockout, etc.) provided that it still functions to convert CO, CO2, and / or H2 into acetyl-CoA.

[0062] An anaerobe is a microorganism that does not require Petition 870200030711, dated 06 / 03 / 2020, page 25 / 61 / 36 oxygen for growth. An anaerobe can react negatively or even die if there is oxygen above a certain threshold. However, some anaerobes can tolerate low levels of oxygen (e.g., 0.000001 to 5% by volume of oxygen). Typically, the microorganism of the invention is an anaerobic microorganism.

[0063] “Acetogens” are obligate anaerobic bacteria that utilize the Wood-Ljungdahl pathway as their primary mechanism for energy conservation and for the synthesis of acetyl-CoA and acetyl-CoA-derived products, such as acetate (Ragsdale, Biochim Biophys Acta, 1784: 1873-1898, 2008). In particular, acetogens utilize the Wood-Ljungdahl pathway as a (1) mechanism for the reductive synthesis of acetyl-CoA from CO2, (2) energy conservation and terminal electron acceptance process, (3) mechanism for CO2 fixation (assimilation) in cellular carbon synthesis (Drake, Acetogenic Prokaryotes, In: The Prokaryotes, 3rd edition, page 354, New York, NY, 2006). All naturally occurring acetogens are C1-fixing, anaerobic, autotrophic, and non-methanotrophic. Typically, the microorganism of the invention is an acetogenic microorganism.

[0064] An “ethanologen” is a microorganism that produces or has the capacity to produce ethanol. Typically, the microorganism of the invention is an “ethanologen.”

[0065] An “autotroph” is a microorganism capable of growing in the absence of organic carbon. Instead, autotrophs utilize inorganic carbon sources, such as CO and / or CO2. Typically, the microorganism of the invention is an autotroph.

[0066] A carboxidotroph is a microorganism capable of using CO₂ as its sole source of carbon and energy. Typically, the microorganism of the invention is a carboxidotroph.

[0067] The microorganism of the invention can be cultivated with the Petition 870200030711, dated 06 / 03 / 2020, page 26 / 61 / 36 gas stream to produce one or more products. For example, the microorganism of the invention can produce, or can be genetically modified to produce, ethanol (WO 2007 / 117157), acetate (WO 2007 / 117157), butanol (WO 2008 / 115080 and WO 2012 / 053905), butyrate (WO 2008 / 115080), 2,3-butanediol (WO 2009 / 151342 and WO 2016 / 094334), lactate (WO 2011 / 112103), butene (WO 2012 / 024522), butadiene (WO 2012 / 024522), methyl ethyl ketone (2-butanone) (WO 2012 / 024522 and WO 2013 / 185123), ethylene (WO 2012 / 026833), acetone (WO 2012 / 115527), isopropanol (WO 2012 / 115527), lipids (WO 2013 / 036147), 3-hydroxypropionate (3-HP) (WO 2013 / 180581), terpenes, including isoprene (WO 2013 / 180584), fatty acids (WO 2013 / 191567), 2-butanol (WO 2013 / 185123), 1,2-propanediol (WO 2014 / 036152), 1-propanol (WO 2014 / 0369152), chorismate-derived products (WO 2016 / 191625), 3-hydroxybutyrate (WO 2017 / 066498) and 1,3-butanediol (WO 2017 / 0066498).In addition to one or more target products, the microorganism of the invention can also produce ethanol, acetate, and / or 2,3-butanediol. In certain embodiments, the microbial biomass itself can be considered a product. These products can be further converted to produce at least one component of diesel, aviation fuel, and / or gasoline. Furthermore, the microbial biomass can be further processed to produce a single-cell protein (SCP).

[0068] A single-cell protein (SCP) refers to a microbial biomass that can be used in protein-rich human and / or animal feeds, often replacing conventional protein supplementation sources such as soybean meal or fishmeal. To produce a single-cell protein or other product, the process may involve separation, processing, or additional treatment steps. For example, the method may involve sterilizing the microbial biomass, centrifuging the microbial biomass, and / or drying the microbial biomass. In Petition 870200030711, dated 06 / 03 / 2020, page 27 / 61 / 36 In certain modalities, 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 ingestion of a diet high in nucleic acid may result in the accumulation of nucleic acid degradation products and / or gastrointestinal discomfort. Single-cell protein may be suitable for feeding animals such as livestock or pets. In particular, animal feed may be suitable for feeding one or more beef cattle, cattle, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo / bison, llamas, alpacas, reindeer, camels, bantengues, jays, yaks, chickens, turkeys, ducks, geese, quail, guinea fowl, pigeons, fish, shrimp, crustaceans, cats, dogs and rodents.The composition of animal feed can be adapted to the nutritional needs of different animals. Furthermore, the process may involve mixing or combining microbial biomass with one or more excipients.

[0069] An excipient may refer to any substance that can be added to microbial biomass to enhance or alter the form, properties, or nutritional content of animal feed. For example, the excipient may comprise one or more of a carbohydrate, fiber, fat, protein, vitamin, mineral, water, flavoring, sweetener, antioxidant, enzyme, preservative, probiotic, or antibiotic. In some embodiments, the excipient may be hay, straw, silage, grains, oils or fats, or other plant material. The excipient may be any feed ingredient identified in Chiba, Section 18: Diet Formulation and Common Feed Ingredients, Animal Nutrition Handbook, 3rd revision, pages 575 to 633, 2014.

[0070] A “natural product” is a product produced by a non-genetically modified microorganism. For example, ethanol, acetate and 2,3 Petition 870200030711, dated 06 / 03 / 2020, page 28 / 61 / 36 butanediol are native products of Clostridium autoethanogenum, Clostridium ljungdahlii and Clostridium ragsdalei. A non-native product is a product produced by a genetically modified microorganism, but is not produced by a non-genetically modified microorganism from which the genetically modified microorganism is derived.

[0071] “Selectivity” refers to the ratio between the production of a target product and the production of all fermentation products produced by a microorganism. The microorganism of the invention can be modified to produce products with a given selectivity or with a minimum selectivity. In one embodiment, a target product accounts for at least about 5% by weight, 10% by weight, 15% by weight, 20% by weight, 30% by weight, 50% by weight, 75% by weight, or 90% by weight of all fermentation products produced by the microorganism of the invention. In another embodiment, the target product accounts for at least 10% by weight of all fermentation products produced by the microorganism of the invention, such that the microorganism of the invention has a selectivity for the target product of at least 10% by weight.In another embodiment, the target product accounts for at least 30% by weight of all fermentation products produced by the microorganism of the invention, such that the microorganism of the invention has a selectivity for the target product of at least 30% by weight. In another embodiment, the target product accounts for at least 90% by weight of all fermentation products produced by the microorganisms, such that the microorganism of the invention has a selectivity for the target product of at least 90% by weight.

[0072] The target products can 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 removal, phase separation and Petition 870200030711, dated 06 / 03 / 2020, page 29 / 61 / 36 extractive fermentation, including, for example, liquid-liquid extraction. In certain embodiments, the target products are recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating the microbial cells from the broth (conveniently by filtration) and recovering one or more target products from the broth. Alcohols and / or acetone can be recovered, for example, by distillation. Acids can be recovered, for example, by adsorption on activated carbon. The separated microbial cells are preferably returned to the bioreactor. The cell-free permeate remaining after removal of the target products is also preferably returned to the bioreactor. Additional nutrients (such as B vitamins) can be added to the cell-free permeate to replenish the medium before it is returned to the bioreactor.

[0073] Culture / fermentation must be carried out under appropriate conditions for the production of the target product. Typically, culture / fermentation is carried out under anaerobic conditions. Reaction conditions to be considered include pressure (or partial pressure), temperature, gas flow rate, liquid flow rate, pH of the media, redox potential of the media, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum substrate gas concentrations to ensure that this gas in the liquid phase does not become limiting, and maximum product concentrations to avoid product inhibition. In particular, the rate of substrate introduction can be controlled to ensure that the gas concentration in the liquid phase does not become limiting, since products can be consumed by the culture under gas-limited conditions.

[0074] Operating a bioreactor at high pressures allows for an increase in the mass transfer rate of gas from the gas phase to the liquid phase. Consequently, it is generally preferable to carry out the culture / fermentation at Petition 870200030711, dated 06 / 03 / 2020, page 30 / 61 / 36 pressures higher than atmospheric pressure. Furthermore, since a given gas conversion rate is, in part, a function of the substrate retention time, and the retention time dictates the required volume of a bioreactor, the use of pressurized systems can considerably reduce the required bioreactor volume and, consequently, the capital cost of the culture / fermentation equipment. This, in turn, means that the retention time, defined as the volume of liquid in the bioreactor divided by the inlet gas flow rate, can be reduced when bioreactors are maintained at elevated pressure instead of atmospheric pressure. Ideal reaction conditions will depend partly on the specific microorganism used. However, in general, it is preferable to operate fermentation at a pressure higher than atmospheric pressure.Furthermore, 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 considerably reduce the required bioreactor volume and, consequently, the capital cost of the fermentation equipment. DESCRIPTION

[0075] Controlling the composition of the C1-containing gaseous substrate supplied to an inoculator and / or bioreactor has been considered particularly useful for promoting cell growth, product selectivity, and stability in both the inoculation reactor and subsequent bioreactors. Preferably, the C1-containing substrate is composition-controlled before being fed to an inoculation reactor to produce an inoculum to feed one or more downstream reactors. Preferably, the inoculation reactor comprises a culture of one or more C1-fixing microorganisms in a liquid nutrient medium and has the capacity to receive the composition-controlled C1-containing gaseous substrate to produce an inoculum via fermentation. Petition 870200030711, dated 06 / 03 / 2020, page 31 / 61 / 36

[0076] The inventors found that when a gaseous substrate containing hydrogen-rich C1 is used for fermentation, the fermentation process generally lacks selectivity and long-term stability. Surprisingly, the inventors found that operating a fermentation process under hydrogen-rich conditions by supplying an alternating stream containing carbon monoxide (CO)-rich C1 to the inoculation reactor results not only in increased biomass growth, but also in greater selectivity for ethanol and greater stability in downstream bioreactors.

[0077] This invention has particular applicability for fermentation processes using industrial gas streams comprising H2 at an H2:CO molar ratio of at least 3:1, however, the invention is also considered beneficial for industrial streams comprising compositions with low H2 content, such as gas streams having H2:CO molar ratios of 2:1 or 1.5:1 or 1.1:1.In one embodiment, the invention provides a process for producing one or more fermentation processes, wherein the process comprises: (a) passing at least a portion of a gaseous substrate containing C1 to an inoculation reactor and at least a portion of the gaseous substrate containing C1 to a bioreactor; (b) fermenting the gaseous substrate containing C1 in the inoculation reactor to produce an inoculum; (c) passing at least a portion of the inoculum to at least one bioreactor; and (d) fermenting the gaseous substrate containing C1 in the bioreactor to produce at least one fermentation product; wherein the gaseous substrate containing C1 that is passed to the inoculation reactor is subjected to at least one H2 removal process before being passed to the inoculation reactor.

[0078] In particular embodiments, the bioreactor comprises one or more primary reactors connected to one or more secondary reactors. In Petition 870200030711, dated 06 / 03 / 2020, p. 32 / 61 / 36 In certain embodiments, the primary reactor (or primary reactors) operates under conditions to promote biomass production, and the secondary reactor (or secondary reactors) operates under conditions to promote metabolite production. In several embodiments, the gaseous substrate containing C1 rich in H2 supplied to the primary and secondary reactors comes from the same industrial source and has substantially the same composition.

[0079] In one embodiment, the gaseous substrate containing CO-rich C1 and the gaseous substrate containing H2-rich C1 are derived from the same industrial source. In several embodiments, at least a portion of a gaseous substrate containing H2-rich C1 is passed through a hydrogen removal process before being fed to the inoculation reactor, where the hydrogen removal process is configured to separate at least a portion of hydrogen from the gaseous substrate containing H2-rich C1 to produce the gaseous substrate containing CO-rich C1. In particular embodiments, the treatment zone comprises a H2 membrane separation module and / or a pressure swing adsorption (PSA) process. Preferably, the hydrogen removal process comprises a membrane separation module.

[0080] In one or more embodiments, the gaseous substrate containing H2-rich C1 is derived from an industrial process.

[0081] In alternative embodiments, the gaseous substrate containing CO-rich C1 comprises a bottled CO gas stream. In one embodiment, the bottled CO gas is mixed with one or more gaseous components, such as nitrogen and / or carbon dioxide. In further embodiments, the gaseous substrate containing CO-rich C1 is a CO-rich gas stream derived from a source other than the gaseous substrate containing H2-rich C1. In one embodiment, the gaseous substrate containing CO-rich C1 is derived from a CO2 electrolysis process. Hydrogen Separation Petition 870200030711, dated 06 / 03 / 2020, pages 33 / 61 / 36

[0082] The volume of gas required may, in some cases, make the use of bottled gas prohibitive due to cost. Therefore, it is preferred that the gaseous substrate containing H2-rich C1 be treated to remove at least a portion of hydrogen from the substrate and produce a gaseous substrate containing C1 and rich in CO. Suitable methods for treating a gaseous substrate containing H2-rich C1 may include, but are not limited to, membrane separation technologies and pressure swing adsorption technologies.

[0083] Membrane separation modules provide a relatively simple and low-cost way to remove at least a portion of hydrogen from a gaseous substrate. For example, a reformer synthesis gas with a composition of 72% by volume H2, 14% by volume CO, 7% by volume CO2 and 7% by volume CH4 at a pressure of 25 bara passing through a demonstrative membrane separation module results in a high-pressure CO-rich stream and a low-pressure H2-rich stream. The high-pressure CO-rich stream at 25 bara contains 50% by volume CO, 16% by volume H2, 25% by volume CH4 and 9% by volume CO2. The low-pressure H2-rich stream is reduced to 1 bara and contains 92% by volume H2, 6% by volume CO2 and 1% by volume CO and CH4. The high-pressure CO-rich stream can be supplied to the inoculator as a gaseous substrate containing CO-rich C1.The high-pressure CO₂-rich stream provides the added benefit of not requiring additional compression, thus avoiding the capital cost associated with the add-on compressor unit for the inoculation reactor.

[0084] Pressure swing adsorption process technologies are a more complex and effective way to remove at least a portion of hydrogen from a gaseous substrate. When using a pressure swing adsorption process, the CO-rich stream is at low pressure. Although the use of a pressure swing adsorption process is Petition 870200030711, dated 06 / 03 / 2020, page 34 / 61 / 36. While feasible, the gaseous substrate containing CO-rich C1 may need to be compressed before being fed to the inoculation reactor or any bioreactor, thus increasing the capital cost associated with the inoculation reactor. This can be at least partially offset, however, by considering the fact that the hydrogen stream produced by the pressure-swinging adsorption process is at high pressure and can be sold as a product. CO2 electrolysis

[0085] An alternative method for providing a gaseous substrate containing C1 and rich in CO is through the use of CO2 electrolysis. CO2 electrolysis processes convert a CO2 feedstock into CO and O2. The use of a CO2 electrolysis process to provide a CO-rich stream to the inoculator may be of interest in industrial sites that comprise a CO2-rich stream in addition to an O2-rich stream. Additionally, it is also considered that the waste gas from the inoculation reactor and / or bioreactor system, which is rich in CO2, can be used as feedstock for CO2 electrolysis units.

[0086] Figure 1 shows a schematic flowchart of one embodiment of the invention. A portion of a gaseous substrate containing C1 is passed through piping media 110 to an inoculation reactor 130, where the substrate containing C1 is fermented to produce an inoculum. At least a portion of the inoculum is passed through piping media 131 to the bioreactor system 140, 150, where a portion of the gaseous substrate containing C1 is also passed through piping media 110 to be fermented to produce at least one product 141, 151. The gaseous substrate containing C1 that is passed to the inoculation reactor 130 is subjected to at least one hydrogen removal process 120 before being sent to the inoculation reactor 130. The hydrogen removal process 120 receives the gaseous substrate containing C1 Petition 870200030711, dated 06 / 03 / 2020, page 35 / 61 / 36 through piping means 110 and removes at least a portion of the hydrogen 121 from the gaseous substrate containing C1 to produce a gaseous substrate containing C1 rich in CO, which is supplied to the inoculation reactor 130 through piping means 122.

[0087] Preferably, the gaseous substrate containing C1 that is passed to the inoculation reactor 130 comprises H2 in an H2:CO molar ratio of less than 1:1. In certain embodiments, the gaseous substrate containing C1 that is passed to the inoculation reactor 130 comprises H2 in an H2:CO molar ratio of less than 0.8:1. Preferably, the gaseous substrate containing C1 that is passed to the inoculation reactor 130 comprises H2 in an H2:CO molar ratio of 0.02:1 to 1:1. In several cases, the hydrogen removal process 120 removes at least a portion of hydrogen through the use of at least one membrane separation module. In several cases, the hydrogen removal process 120 removes at least a portion of hydrogen through the use of at least one pressure swing adsorption process.In various embodiments, the hydrogen removal process 120 removes at least a portion of hydrogen through the use of a membrane separation module and a pressure swing adsorption process.

[0088] In certain cases, the gaseous substrate containing C1 that is supplied to the inoculation reactor 130 and the bioreactor system 140, 150 is derived at least in part from an industrial source. Preferably, the industrial source is selected from the group consisting of carbohydrate fermentation, gaseous fermentation, cement manufacturing, paper and pulp manufacturing, steel manufacturing, petroleum refining and associated processes, petrochemical production, coke production, anaerobic or aerobic digestion, synthesis gas, natural gas extraction, petroleum extraction, metallurgical processes, for the production and / or refining of aluminum, copper and / or ferroalloys, geological reservoirs and catalytic processes. Petition 870200030711, dated 06 / 03 / 2020, page 36 / 61 / 36

[0089] Preferably, the fermentation product 141, 151 produced by the bioreactor system 140, 150 is selected from the group consisting of: ethanol, acetate, butanol, butyrate, 2,3-butanediol, 1,3-butanediol, lactate, butene, butadiene, methyl ethyl ketone, ethylene, acetone, isopropanol, lipids, 3-hydroxypropionate, isoprene, fatty acids, 2-butanol, 1,2-propanediol, 1-propanol, monoethylene glycol, isobutene and C6-C14 alcohols. In several cases, at least a portion of the product 141, 151 is further converted into at least one component of diesel fuel, aviation fuel, gasoline, propylene, nylon 6-6, rubber and / or resins. In several cases, at least one fermentation product 141, 151 is microbial biomass. This microbial biomass can, in some cases, be further processed to produce at least one component of animal feed.

[0090] In several embodiments, the fermentation broth from a bioreactor 140 can be passed to another bioreactor 150 within the bioreactor system 140,150 through piping means 142.

[0091] Figure 2 shows a schematic flowchart of one embodiment of the invention. A portion of a gaseous substrate containing C1 is passed through piping means 210 to an inoculation reactor 230, where the substrate containing C1 is fermented to produce an inoculum. At least a portion of the inoculum is passed through piping media 231 to the bioreactor system 240, 250, where a portion of the gaseous substrate containing C1 is also passed through piping media 210 to be fermented to produce at least one product 241, 251. The gaseous substrate containing C1 that is passed to the inoculation reactor 230 and the bioreactor system 140, 150 undergoes at least one hydrogen removal process 220 before being sent to the inoculation reactor 230. The hydrogen removal process 220 receives the gaseous substrate containing C1 through piping media 210 and removes Petition 870200030711, dated 06 / 03 / 2020, page 37 / 61 / 36 at least a portion of the hydrogen 221 from the gaseous substrate containing C1 to produce a gaseous substrate containing C1 rich in CO, which is supplied to the inoculation reactor 230 through piping means 222 and to the bioreactor system, 140,150 through piping means 223.

[0092] Preferably, the gaseous substrate containing C1 that is passed to the inoculation reactor 230 and to the bioreactor system 240,250 comprises H2 at a molar ratio of H2:CO of less than 1:1. In certain embodiments, the gaseous substrate containing C1 that is passed to the inoculation reactor 230 and to the bioreactor system 240,250 comprises H2 at a molar ratio of H2:CO of less than 0.8:1. Preferably, the gaseous substrate containing C1 that is passed to the inoculation reactor 230 and to the bioreactor system 240,250 comprises H2 at a molar ratio of H2:CO between 0.02:1 and 1:1. In several cases, the hydrogen removal process 220 removes at least a portion of hydrogen through the use of at least one membrane separation module. In many cases, the hydrogen 220 removal process removes at least a portion of hydrogen through the use of at least one pressure-swinging adsorption process.In various embodiments, the hydrogen 220 removal process removes at least a portion of hydrogen through the use of a membrane separation module and a pressure swing adsorption process.

[0093] In certain cases, the gaseous substrate containing C1 that is supplied to the inoculation reactor 230 and the bioreactor system 240, 250 is derived, at least in part, from an industrial source. Preferably, the industrial source is selected from the group consisting of carbohydrate fermentation, gaseous fermentation, cement manufacturing, paper and pulp manufacturing, steel manufacturing, petroleum refining and associated processes, petrochemical production, coke production, anaerobic or aerobic digestion, synthesis gas, natural gas extraction, petroleum extraction, processes Petition 870200030711, dated 06 / 03 / 2020, page 38 / 61 / 36 metallurgical, for the production and / or refining of aluminum, copper and / or ferroalloys, geological reservoirs and catalytic processes.

[0094] Preferably, the fermentation product 241, 251 produced by the bioreactor system 240, 250 is selected from the group consisting of: ethanol, acetate, butanol, butyrate, 2,3-butanediol, 1,3-butanediol, lactate, butene, butadiene, methyl ethyl ketone, ethylene, acetone, isopropanol, lipids, 3-hydroxypropionate, isoprene, fatty acids, 2-butanol, 1,2-propanediol, 1-propanol, monoethylene glycol, isobutene and C6-C14 alcohols. In several cases, at least a portion of the product 241, 251 is further converted into at least one component of diesel fuel, aviation fuel, gasoline, propylene, nylon 6-6, rubber and / or resins. In several cases, at least one fermentation product 241, 251 is microbial biomass. This microbial biomass can, in some cases, be further processed to produce at least one component of animal feed.

[0095] In several embodiments, the fermentation broth from a bioreactor 240 can be passed to another bioreactor 250 within the bioreactor system 240,250 through piping means 242.

[0096] Figures 3, 4 and 5 illustrate various embodiments of the invention, using a hydrogen production process from a refining operation as the industrial source of gaseous substrate containing H2-rich C1. A typical hydrogen production process, as depicted in Figure 3, Figure 4 and Figure 5, contains the following stages: (i) a reforming process, in which a feedstock containing CH4 is converted into a synthesis gas stream comprising CO and H2; (ii) at least one water-gas exchange step, in which a portion of the CO is reacted with water to produce H2 and CO2; and (iii) a pressure swing adsorption (PSA) module adapted to recover hydrogen from the gas stream.

[0097] Figure 3 shows an embodiment of the invention that uses a Petition 870200030711, dated 06 / 03 / 2020, page 39 / 61 / 36 gaseous substrate containing C1 rich in H2 from a reforming process 310. At least a portion of the gaseous substrate containing C1 rich in H2 flows to a membrane separation module 350 through piping means 312. The membrane separation module 350 separates the gaseous substrate containing C1 into a high-pressure CO-rich stream and a low-pressure H2-rich stream. At least a portion of the low-pressure CO-rich stream is passed to an inoculation reactor 370 via piping means 352. At least a portion of the low-pressure H2-rich stream is passed to a pressure-swing adsorption process 360 via piping means 351. In at least one embodiment, the gaseous substrate is passed to a compressor before being passed to the pressure-swing adsorption process 360.In one embodiment, the CO-rich stream comprises at least 40% CO, or at least 50% CO, or at least 60% CO. In one embodiment, the pressure of the CO-rich C1-containing stream is at least 15 bar, or at least 20 bar, or at least 25 bar.

[0098] In various embodiments, the process may include several water-gas exchange processes 320, 330 and / or multiple hydrogen removal processes 350, 340, 360. As shown in Figure 3, the gaseous substrate containing C1 may first be passed from a reforming process 310 to a water-gas exchange process 320 via piping means 311 to convert at least a portion of CH4 to a synthesis gas stream comprising CO and H2. This gas stream may optionally pass through one or more water-gas exchange processes 330 via piping means 321 and be fed to one or more hydrogen removal processes 340 to separate at least a portion of the hydrogen 341 from the gas stream. This stream can then be passed through one or more other hydrogen removal processes 360 via piping means 342. The stream from one or more other processes of Petition 870200030711, dated 06 / 03 / 2020, page 40 / 61 / 36 Hydrogen removal 360 can be sent to the bioreactor 380 through piping means 361 for fermentation. At least a portion of the substrate not sent to the bioreactor can optionally be sent to the reforming process 310 through piping means 362. In several cases, the bioreactor 380 receives the gaseous substrate and produces one or more fermentation products 381. Optionally, the waste gas from the inoculation reactor 370 and the bioreactor 380 can be passed back to the reforming process 310 through separate piping means 372, 382 and / or a blended stream 378.

[0099] In the various embodiments, the inoculation reactor 370 and the bioreactor 380 are configured in a stepwise manner, whereby the inoculation reactor 370 ferments a gaseous substrate containing C1 rich in CO to produce an inoculum, which is then supplied to the bioreactor 380 via piping means 371. By using this inoculum in the bioreactor 380, the product selectivity and the stability of the fermentation process are improved.

[00100] In another embodiment, as shown in Figure 4, a C1-containing stream rich in H2 from a reforming process 410 flows to the pressure swing adsorption process 450 through piping means 412 provided upstream of the inoculation reactor 470. The pressure swing adsorption process 450 separates the C1-containing stream into a high-pressure H2-rich stream and a low-pressure CO-rich stream. The low-pressure CO-rich stream may be passed to a compressor before being passed to the inoculation reactor 470 through piping means 452. In one embodiment, the CO-rich stream passed to the inoculation reactor 470 comprises at least 30% CO or at least 40% CO or at least 50% CO or at least 60% CO. The separated hydrogen can be passed from the pressure swing adsorption process (450) to another pressure swing adsorption process (of...). Petition 870200030711, dated 06 / 03 / 2020, page 41 / 61 / 36 pressure 460 through piping means 451. In various embodiments, the process may include several water-gas exchange processes 420, 430 and / or multiple hydrogen removal processes 450, 440, 460.

[00101] As shown in Figure 4, the substrate containing C1 can first be passed from a reforming process 410 to a water-gas exchange process 420 via piping means 411 to convert at least a portion of the CH4 into a synthesis gas stream comprising CO and H2. This gas stream can then be passed to one or more water-gas exchange processes 430 via piping means 421 and fed to one or more hydrogen removal processes 440 via piping means 431 to separate at least a portion of the hydrogen 441 from the gas stream. This stream can then be passed to one or more other hydrogen removal processes 460 via piping means 442. The stream from one or more other hydrogen removal processes 460 can be sent to the bioreactor 480 via piping means 461 for fermentation.At least a portion of the substrate not sent to the bioreactor may optionally be sent to the reforming process 410 via piping means 462. In several cases, the bioreactor 480 receives the gaseous substrate and produces one or more fermentation products 481. Optionally, the waste gas from the inoculation reactor 470 and the bioreactor 480 may be passed back to the reforming process 410 via separate piping means 472, 482 and / or a blended stream 478.

[00102] In various embodiments, the inoculation reactor 470 and the bioreactor 480 are configured in a stepwise manner, whereby the inoculation reactor 470 ferments a gaseous substrate containing CO-rich C1 to produce an inoculum, which is then supplied to the bioreactor 480 via piping means 471. By using this inoculum in the bioreactor 480, the product selectivity and stability of the fermentation process are improved. Petition 870200030711, dated 06 / 03 / 2020, pages 42 / 61 / 36 are improved.

[00103] In another embodiment, as shown in Figure 5, the C1-containing stream from the reforming process 510 can be sent to multiple hydrogen removal processes 540, 550, 560, 590 before being sent to the inoculation reactor 570 and / or to the bioreactor 580. In several cases, the C1-containing stream can be sent to a compressor before and / or between a hydrogen removal process. By sending the C1-containing stream to multiple hydrogen removal processes, the CO composition in the C1-containing stream can be further enriched.

[00104] In various embodiments, the process may include multiple water-gas exchange processes 520, 530 in combination with multiple hydrogen removal processes 540, 550, 560. As shown in Figure 5, the gaseous substrate containing C1 may first be passed from a reforming process 510 to a water-gas exchange process 520 via piping means 511 to convert at least a portion of the CH4 into a synthesis gas stream comprising CO and H2. This gas stream may then be passed to one or more water-gas exchange processes 530 via piping means 521 and fed to one or more hydrogen removal processes 540 via piping means 531 to separate at least a portion of the hydrogen 541 from the gas stream. This stream may then be passed to one or more other hydrogen removal processes 560 via piping means 542.The stream from one or more other hydrogen removal processes 560 can be sent to the bioreactor 580 through piping means 561 for fermentation. At least a portion of the substrate not sent to the bioreactor can optionally be sent to a subsequent hydrogen removal process 550 through piping means 562 and optionally to an additional hydrogen removal process 590 through piping means 551, which can finally be sent to the inoculation reactor. Petition 870200030711, dated 06 / 03 / 2020, page 43 / 61 / 36 570, through piping means 591, to produce an inoculum.

[00105] In several cases, the bioreactor 580 receives the gaseous substrate and produces one or more fermentation products 581. Optionally, the waste gas from the inoculation reactor 570 and the bioreactor 580 can be passed back to the reforming process 510 through separate piping means 572, 582 and / or a blended stream 578.

[00106] In the various embodiments, the inoculation reactor 570 and the bioreactor 580 are configured in a stepwise manner, whereby the inoculation reactor 570 ferments a gaseous substrate containing CO-rich C1 to produce an inoculum, which is then supplied to the bioreactor 580 via piping means 571. By using this inoculum in the bioreactor 580, the product selectivity and stability of the fermentation process are improved.

[00107] It should be understood that, although Figure 3, Figure 4 and Figure 5 are representations of an integration with a hydrogen production process, the actual application should not be limited to integration with a hydrogen production process. EXAMPLES

[00108] The following examples illustrate the invention in more detail, but they should certainly not be interpreted as limiting its scope in any way. EXAMPLE 1

[00109] This example demonstrates the comparative performance of two reactors equipped with a gaseous substrate comprising 68% by volume of H2, 3.8% by volume of CO, 26% by volume of CO2 and 1% by volume of N2, a molar ratio of 18:1 of H2:CO. The only difference between the operating parameters of the two reactors was the conditions under which the inoculum was produced for each reactor. Figure 6a and Figure 6b show metabolite and gas profiles in a first bioreactor that received the produced inoculum. Petition 870200030711, dated 06 / 03 / 2020, page 44 / 61 / 36 under CO-rich conditions. Figure 7a and Figure 7b show metabolite and gas profiles in a second bioreactor that received the inoculum produced under H2-rich conditions. Both reactors consume H2, CO, and CO2 with similar efficiency, but the reactor receiving an inoculum from an H2-rich inoculation reactor (Figure 7a) reduced the selectivity for ethanol. EXAMPLE 2

[00110] This example demonstrates the comparative performance of inoculated reactors from inoculation reactors operated under different gas conditions. Figure 8a and Figure 8b show the metabolite and gas profiles of a fermentation inoculated with a culture received from an inoculation produced with the following gas composition: 48% by volume H2, 40% by volume CO, 2% by volume CO2, and 10% by volume N2. Figure 9a and Figure 9b illustrate the metabolite and gas profiles of a fermentation inoculated with a culture received from an inoculation produced under CO-rich conditions. The ethanol selectivity demonstrated by the reactor fed from the CO-rich gas inoculation reactor (Figure 9a) is much higher than that of the reactor that received an inoculum from an H2-rich inoculation reactor (Figure 8a).

[00111] All references, including publications, patent applications and patents, cited in this document are incorporated herein by reference to the extent that each reference has been individually and specifically indicated for incorporation by reference and has been presented herein in its entirety. Reference to any prior art in this descriptive report is not and should not be construed as an acknowledgment that the prior art forms part of the common general knowledge in the field of entrepreneurship in any country.

[00112] The use of the terms “a” and “an”, “the”, “the” and similar referents in the context of the invention description (especially in the context Petition 870200030711, dated 06 / 03 / 2020, p. 45 / 61 / 36 of the attached claims) should be interpreted to cover both the singular and the plural, unless otherwise indicated or the context clearly dictates otherwise. The terms “comprising (or including)”, “having (or having)”, “including (or including)” and “containing (or containing)” should be interpreted as open terms (i.e., meaning “including, but without limitation”), unless otherwise indicated. The term “consisting (or 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 innovative characteristics of the composition, process or method. The meaning of the use of the alternative (e.g., “or”) should be understood as one, both, or any combination of the alternatives.As used in this document, the term “approximately” means ± 20% of the indicated range, value, or structure, unless otherwise indicated.

[00113] The mention of value ranges in this document is merely intended to serve as a shorthand method for referring individually to each separate value within the range, unless otherwise indicated in this document, and each separate value is incorporated into the descriptive report as if it were individually mentioned in this document. For example, any concentration range, percentage range, ratio range, whole number range, size range, or thickness range should be understood as including the value of any whole number within the stated range and, where appropriate, its fractions (such as one-tenth and one-hundredth of a whole number), unless otherwise indicated.

[00114] All methods described in this document may be performed in any appropriate order, unless otherwise indicated in this document or the context clearly dictates otherwise. Petition 870200030711, dated 06 / 03 / 2020, pp. 46 / 61 / 36 The use of any and all examples, or illustrative language (e.g., “such as”) provided in this document is solely for the purpose of better clarifying the invention and does not represent a limitation of the scope of the invention, unless otherwise claimed. No language in the descriptive report should be interpreted as indicating any unclaimed element as essential to the practice of the invention.

[00115] Preferred embodiments of this invention are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art after reading the above description. The inventors expect that those skilled in the art will employ such variations as appropriate, and the inventors intend that the invention be practiced in a manner other than that specifically described herein. Consequently, this invention includes all modifications and equivalents of the subject matter mentioned in the appended claims, as permitted by applicable law. Furthermore, any combination of the elements described above in all possible variations is encompassed by the invention, unless otherwise indicated herein or the context clearly dictates otherwise. Petition 870200030711, dated 06 / 03 / 2020, pages 47 / 61

Claims

1 / 2 CLAIMS 1. A process for producing one or more fermentation products, wherein the process is characterized in that it comprises: a. supplying a gaseous substrate containing CO₂-rich C₁ to an inoculation reactor comprising a liquid nutrient medium containing a culture of one or more C₁-fixing microorganisms; b. fermenting the gaseous substrate containing CO₂-rich C₁ to produce an inoculum; c. passing at least a portion of the inoculum to a bioreactor system, wherein the bioreactor system comprises at least one bioreactor containing a culture of one or more C₁-fixing microorganisms in a liquid nutrient medium; d. passing a gaseous substrate containing H₂-rich C₁ to the bioreactor system containing C₁; and e.to ferment the gaseous substrate containing C1 rich in H2 to produce at least one fermentation product; wherein the gaseous substrate containing C1 rich in CO comprises H2 in a molar ratio of H2:CO of less than 1:1; and wherein the gaseous substrate containing C1 rich in H2 comprises H2 in a molar ratio of H2:CO of at least 1.1:

1.

2. Process according to claim 1, characterized in that the gaseous substrate containing CO-rich C1 comprises H2 at a molar ratio of H2:CO of less than 0.5:

1.

3. Process according to claim 1, characterized in that the gaseous substrate containing CO-rich C1 comprises H2 in a molar ratio of H2:CO between 0.02:1 and 1:

1.

4. Process according to claim 1, characterized in that the gaseous substrate containing H2-rich C1 comprises H2 in a molar ratio of H2:CO between 1.1:1 and 6:

1. Petition 870260055395, dated 08 / 06 / 2026, page 11 / 14 2 / 2 5. Process according to claim 1, characterized in that the C1-fixing microorganism is a carboxidotrophic bacterium.

6. Process according to claim 5, characterized in that the carboxidotrophic bacteria is selected from the group consisting of Moorella, Clostridium, Ruminococcus, Acetobacterium, Eubacterium, Butyribacterium, Oxobacter, Methanosarcina and Desulfotomaculum.

7. Process according to claim 5, characterized in that the carboxidotrophic bacterium is Clostridium autoethanogenum.

8. Process according to claim 1, characterized in that the bioreactor system comprises one or more primary bioreactors connected to one or more secondary bioreactors. Petition 870260055395, dated 08 / 06 / 2026, p. 12 / 14

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