Method for producing biomass using hydrogen-oxidizing bacteria

Abstract

The present invention provides a method for producing biomass comprising at least 65% protein from hydrogen-oxidizing microorganisms using one or more input streams comprising one or more gaseous substrates and a nutrient composition, the input streams and nutrient composition being controlled, and wherein biomass is produced at a rate greater than 10 g / l / day. The produced and isolated biomass can be used as feed or provide nutrition to one or more organisms.

Description

[0001] This application is a divisional application of PCT invention patent application No. PCT / EP2021 / 060880 filed on April 26, 2021 (which entered the Chinese national phase on October 24, 2022, with national phase application No. 202180030748.4 and invention title "Method for producing biomass using hydrogen-oxidizing bacteria"). Technical Field

[0002] The present invention provides a method for producing biomass containing at least 65% protein from hydrogen-oxidizing microorganisms using one or more input streams containing one or more gaseous substrates. Background Technology

[0003] There is a need to produce protein as a food source in a more sustainable way to reduce resource use and greenhouse gas (GHG) emissions. The growing global population is placing increasing pressure on resource and environmental availability. Animal products such as meat, fish, milk, and eggs are important dietary sources of protein, but livestock use vast amounts of agricultural land, energy, and water. This is largely due to the fact that animal feed consists of large quantities of plants grown specifically for this purpose. An alternative source of protein components for animal feed, or even for direct human consumption, is microbial protein, which can be tailored to specific nutritional needs. Microbial protein is considered a highly sustainable protein source due to the efficiency of land, energy, and water use in its production.

[0004] Many industrial processes generate waste gas streams containing carbon dioxide and other gaseous components. Examples include energy production through combustion, lime production, fertilizer production, and cement production, which are major sources of atmospheric carbon dioxide and other GHGs. Carbon oxides from industrial sources are primarily produced by the combustion of fossil fuels and / or chemicals and are classified as GHGs due to their contribution to harmful environmental conditions. Other industrial processes involving waste combustion include municipal solid waste, sewage sludge, plastics, tires, agricultural residues, and coal-fired or gas-fired power plants.

[0005] Microorganisms require carbon sources to survive, grow, and produce chemical products. Therefore, carbon oxides derived from industrial gaseous effluents represent a potentially inexpensive, sustainable, and scalable way to obtain carbon for microbial-mediated production of protein-rich foods, as well as a method to reduce the amount of carbon dioxide released directly into the atmosphere.

[0006] Therefore, there is a desire to convert gaseous feedstocks into sources of high-quality biomass. Consequently, there is a need for improved, simple, high-yield, and economical methods for biomass production that can be used, for example, as animal feed or for direct human consumption.

[0007] Previous work has been known to involve certain applications of chemoautotrophic microorganisms in the capture and conversion of carbon dioxide gas into fixed carbon. However, many of these methods have drawbacks that limit the effectiveness, economic feasibility, practicality, and commercial adoption of the described methods. In particular, achieving maximum production rates in a continuous and stable manner (preferably by maintaining high concentrations of microorganisms in the liquid phase of the bioreactor) while maintaining high protein content is a challenge without making the method economically unattractive.

[0008] Patent US9157058B2 (WO2013090769A2) describes apparatus and methods for growing and maintaining microorganisms and / or biological processes using one or more gases as electron donors, electron acceptors, carbon sources, or other nutrients, and for biological processes that convert hydrogen and carbon dioxide, or syngas, or producer gas, into lipid products, bio-based oils, or other biochemical products. However, it does not disclose optimal conditions for microbial growth and maintenance, nor does it disclose methods for optimizing the protein production capacity of microorganisms.

[0009] Patent US9206451B2 describes a system and method for using chemoautotrophic microorganisms to capture carbon from industrial waste, but it does not disclose controlled optimal process conditions for growth and maintenance, nor does it disclose preferred microorganisms or methods for optimizing the protein production capacity of the microorganisms.

[0010] Patent application WO2018144965A1 describes microorganisms and bioprocesses for converting gaseous substrates (e.g., renewable hydrogen and waste carbon dioxide generator gas or syngas) into high-protein biomass. However, it does not disclose controlled optimal process conditions for the growth and maintenance of microorganisms, nor does it disclose methods for optimizing the protein production capacity of microorganisms.

[0011] Patent application WO2019010116A1 describes a method for producing multicarbon compounds from simple gaseous feedstocks such as carbon dioxide, hydrogen, and oxygen by culturing aggregates of microbial cells specifically selected for this purpose in an aqueous culture medium. However, it does not disclose controlled optimal process conditions for the growth and maintenance of the microorganisms, nor does it disclose methods for optimizing the protein production capacity of the microorganisms.

[0012] TG Volova and VA Barashkov described a method for culturing hydrogen-oxidizing bacteria to produce biomass with a dry weight protein content of 64% to 76% in “Characteristics of Proteins Synthesized by Hydrogen-Oxidizing Microorganisms” (Applied Biochemistry and Microbiology, 2010), but did not disclose the amounts of carbon dioxide, hydrogen, and oxygen used, nor did they disclose methods for optimizing productivity.

[0013] Patent application US2010120104A1 discloses a multi-step method for producing biomass by capturing carbon via obligate and / or facultative chemoautotrophic microorganisms and / or cell extracts containing enzymes from chemoautotrophic microorganisms. Various electron donors and acceptors, as well as microorganisms, are disclosed for the method, but specific process parameters are not described.

[0014] Patent application WO2011139804A2 discloses a method for producing biomass by capturing carbon with one carbon atom through hydroxyl microorganisms, and a suitable bioreactor utilizing hydrogen and oxygen, wherein the volume of the gas occupies at least about 2% of the total volume of the column in which the gas is located. Specific process parameters for optimizing protein production capacity are not described.

[0015] Patent application WO2017165244A1 discloses a method for producing biomass by capturing and transforming inorganic and / or organic molecules containing only one carbon atom through chemoautotrophic microorganisms. It discloses the growth of *Cupriavidus necator* in a standard, off-the-shelf, laboratory-scale bioreactor using H2 and CO2 to achieve a dry biomass density exceeding 40 g / L within 6 days. It also discloses that *Cupriavidus necator* strains DSM 531 and DSM 541, grown in liquid MSM medium with an unspecified Knallgas mixture as the sole carbon and energy source, accumulated over 70% and 80% of total protein by weight, respectively, in samples collected during the arithmetic growth phase. However, the disclosure indicates that *Cupriavidus necator* is grown under limited oxygen conditions, which is not optimal for producing biomass with high protein concentrations at high industrial rates. Furthermore, the disclosed system for growing *Cupriavidus necator* is a continuously fed batch system, where the specific growth rate is a function of an undisclosed gas transfer rate. Therefore, WO2017165244A1 does not disclose the ability to produce biomass at a high rate, wherein the biomass contains a high protein content, nor does it disclose the amounts of carbon dioxide, hydrogen, and oxygen used to obtain biomass with a high protein content and / or a high biomass production rate.

[0016] Morinaga et al. described the conditions for culturing *Alcaligenes eutrophus* in "Growth Characteristics and Cell Composition of *Alcaligenes eutrophus* in Chemostat Culture" (Agric. Biol. Chem., 1977), but they did not disclose how to achieve biomass production at a rate greater than approximately 7.2 g / L / day and a protein content greater than 65%, nor did they disclose the growth medium capable of supporting high concentrations of microorganisms. Therefore, combining high productivity with high-density microorganisms remains a challenge.

[0017] Therefore, it remains necessary to identify a group of chemoautotrophic microorganisms that can be grown in novel or conventional, controlled, and scalable controlled reaction vessels to produce proteins and other nutritionally beneficial products in a stable and commercially viable manner with high productivity. The growth and maintenance of these microorganisms should then be managed through controlled optimal process conditions to fine-tune their metabolic and physiological characteristics, ultimately resulting in high yields of high-quality biomass with high protein content, preferably by maintaining high concentrations of microorganisms in the liquid phase of the bioreactor. Summary of the Invention

[0018] This invention provides a commercially viable method for producing high-quality biomass with high protein content at high productivity. This is achieved by using an input stream comprising gaseous carbon and energy managed under controlled optimal process conditions, a nutrient supply managed under controlled optimal process conditions, and a culture of chemoautotrophic hydrogen oxidizing microorganisms. This solution reduces and optimizes substrate limitations and enables higher oxygen utilization, thereby enabling increased productivity, preferably in a manner that maintains safe operating conditions within the bioreactor, which can be achieved by mitigating potentially explosive gas mixtures.

[0019] Therefore, the object of the present invention is to provide a method for producing biomass from hydrogen-oxidizing microorganisms using one or more input streams containing one or more gaseous substrates, said biomass comprising at least 65% protein of total biomass on a dry weight basis, said gaseous substrates comprising hydrogen and / or oxygen and / or carbon dioxide, said method comprising contacting microorganisms in a liquid phase with a nutrient composition comprising compounds containing carbon and / or nitrogen and / or phosphorus and the gaseous substrates, wherein the input streams and nutrient compositions are controlled and wherein biomass is produced at a rate greater than 10 g / L / day.

[0020] Another objective is to provide a method in which controlling the input flow includes maintaining a molar ratio of hydrogen:oxygen:carbon dioxide dissolved in the liquid phase at a distance of 0 to 500 mm, preferably 0 to 100 mm, from the gas phase in direct contact with the liquid phase at a distance of 0 to 12.748:0 to 4.25:0 to 2.0.

[0021] Another objective is to provide a method for producing biomass from bacteria selected from the genus *Alcaligines*.

[0022] Another objective is to provide a method for producing biomass from bacteria selected from the genus Cupriavidus sp.

[0023] In another aspect, the present invention provides a method for separating produced biomass and removing nutrient compositions, the method comprising downstream processing.

[0024] Another objective is to provide a method for separating produced biomass by removing a nutrient composition, the method comprising dehydrating and / or drying the biomass such that the biomass contains less than 5% by weight of water.

[0025] Another object is to provide a biomass containing protein, said protein comprising the following amino acid contents: 0.9% to 4.8% histidine of total biomass dry weight protein content, 2.0% to 6.9% isoleucine of total biomass dry weight protein content, 3.8% to 12.0% leucine of total biomass dry weight protein content, 3.0% to 11.1% lysine of total biomass dry weight protein content, 1.1% to 5.4% methionine of total biomass dry weight protein content, 1.7% to 8.5% phenylalanine of total biomass dry weight protein content, 1.6% to 6.9% threonine of total biomass dry weight protein content, 0.4% to 3.9% tryptophan of total biomass dry weight protein content, and 1.7% to 9.3% valine of total biomass dry weight protein content.

[0026] In another aspect, the present invention provides a biomass comprising 2.3% to 18% of a lipid content of total biomass dry weight, the lipid content comprising the following fatty acid contents: 23% to 60% of a C16:0 palmitic acid content of total biomass dry weight fatty acid content, 3.8% to 22.3% of a C16:1 palmitoleic acid content of total biomass dry weight fatty acid content, and 23% to 60% of a C17:1 heptadecanoic acid content of total biomass dry weight fatty acid content.

[0027] Another objective is to provide the use of nutrient compositions obtained by means of separating biomass produced as nutrient compositions for the production of biomass.

[0028] Another object of the present invention is to provide the use of biomass, wherein biomass is used to feed one or more organisms or to provide nutrition to one or more organisms. Attached Figure Description

[0029] Figure 1 A preferred embodiment of a system for running the method is disclosed.

[0030] Figure 2 Examples are shown of the range of total protein content and essential amino acid amounts per 100g of isolated biomass produced according to the method of the present invention.

[0031] Figure 3 An example is shown of the proportion of the most abundant fatty acid in the total fatty acid content of isolated biomass produced according to the method of the present invention.

[0032] Figure 4 Examples are shown of the range of total protein content and essential amino acid content per 100g of isolated biomass produced according to the method of the invention, compared to the range of essential amino acid amounts in typical soybean meal and fishmeal used in animal agriculture.

[0033] Figure 5 shows the effects of different oxygen ( Figure 5A ) and hydrogen ( Figure 5B Prediction of biomass production rate at the input concentration percentage.

[0034] Figure 6 The invention illustrates a prediction of the protein content, by dry weight, of total biomass from hydrogen-oxidizing microorganisms produced according to the present invention, wherein the controlled input stream comprises the addition of a certain molar ratio of hydrogen to oxygen in the liquid phase.

[0035] Figure 7 The invention illustrates a prediction of the protein content by dry weight of total biomass from hydrogen-oxidizing microorganisms produced according to the invention, wherein the control input stream includes a control ratio growth rate.

[0036] Figure 8A A prediction of the preferred hydrogen transfer rate related to the biomass production rate produced according to the present invention is shown.

[0037] Figure 8B A prediction of the preferred oxygen transfer rate associated with the biomass production rate produced according to the present invention is shown.

[0038] The following list includes definitions of the reference numerals used in the accompanying figures:

[0039] 1. Oxygen / Air Input

[0040] 2 Hydrogen input

[0041] 3. Carbon dioxide input

[0042] 4. Addition of inorganic nitrogen (e.g., urea)

[0043] 5 pH buffer added

[0044] 6. Liquid growth medium added

[0045] 7 Unused gas recirculation

[0046] 8. Removal of liquids containing biomass

[0047] 9. Downstream treatment steps 10. Liquid recirculation

[0048] 11 Optional downstream processing steps Detailed Implementation

[0049] definition

[0050] In this article, biomass is understood to mean the total weight of microorganisms and their offspring, products and / or metabolites.

[0051] In this paper, a bioreactor is understood as a system for maintaining and / or growing microorganisms, which includes a gas phase, commonly referred to as the headspace, and a liquid phase. Microorganisms grow and are maintained in the liquid phase.

[0052] Hydrogen-oxidizing microorganisms are intended to be understood as facultative chemoautotrophic bacteria that can use hydrogen as an electron donor. The group of aerobic hydrogen-oxidizing bacteria (also known as Knallgas bacteria) is physiologically defined and includes bacteria from different taxonomic units. This group is defined by its ability to use gaseous hydrogen as an electron donor and oxygen as an electron acceptor, and to fix carbon dioxide.

[0053] In this paper, input flow is understood to mean the supply of nutrients and / or energy, including liquid and / or gas phases, for the growth and / or maintenance of microorganisms.

[0054] In this article, the liquid phase is understood to refer to the volume containing liquid material. Microorganisms typically grow and are maintained in the liquid phase. Biomass is primarily located within the liquid phase. The liquid phase may also contain solid material used as a growth and maintenance substrate for microbial attachment.

[0055] The liquid phase may contain compounds containing carbon, nitrogen, and / or phosphorus, wherein the carbon-containing compounds may be formate or methanol, but are preferably understood to be substantially limited to dissolved CO2 or urea, the latter of which may also be considered a bioavailable source of N2. Formate or methanol can be converted into CO2 in the liquid phase of the bioreactor, which may be indirectly supplied as a gaseous substrate, for example, by enzymes present inside or outside the microorganisms.

[0056] In this article, the gas phase is understood to refer to the volume composed of gaseous material and in contact with the liquid phase. In a bioreactor, the gas phase is often referred to as the headspace and is typically located directly above the liquid phase. For clarity, the gas or gaseous substrate bubbled into the liquid phase is not part of the gas phase but becomes part of it upon leaving the liquid phase.

[0057] Gaseous substrates are understood to refer to the gaseous supply of nutrients and / or energy used for the growth and / or maintenance of microorganisms.

[0058] A cement kiln is understood to refer to the space used for the high-temperature processing stage of the manufacture of Portland and other types of hydraulic cement, in which calcium carbonate reacts with silica-containing minerals to form a mixture of calcium silicates.

[0059] In this article, syngas or synthesis gas is understood to refer to a mixture containing carbon monoxide, carbon dioxide, and hydrogen. Syngas is produced by gasifying carbon-containing fuels into gaseous products. The exact chemical composition of syngas varies depending on the raw materials and processes used. One use of syngas is as a fuel for producing steam or electricity. Another use is as a basic chemical building block in many petrochemical and refining processes. Syngas can be produced from many sources, including natural gas, coal, petroleum-based materials, biomass, other materials that would be disposed of as waste, or virtually any hydrocarbon feedstock.

[0060] "Knallgas" is understood to refer to a mixture of highly flammable hydrogen and oxygen. A molar ratio of 2:1 is sufficient for maximum ignition efficiency.

[0061] Bubbling is understood as the process by which gas bubbles as it passes through a liquid.

[0062] In this document, the dry weight or dry matter of a material is understood to mean a material that consists of all its components but contains essentially no water. Examples of obtaining the dry weight or dry matter of a material include centrifugation, drum drying, belt drying, evaporation, freeze drying, heating, spray drying, vacuum drying, and / or vacuum filtration to remove the water content of the material.

[0063] Downstream treatment is understood to mean one or more treatment steps applied to the liquid phase removed from the bioreactor, which may include a kill process, a dehydration process, or a drying process.

[0064] The killing step is understood as the process of achieving the reproductive inactivation of microorganisms. This process can occur in the liquid phase, dehydrated liquid phase, or dried biomass, for example, by using ultra-high pressure homogenizers, acids, alkalis, solvents, or heat-based microbial killing methods.

[0065] In this document, dehydration is understood as the first process of removing liquid and / or nutrient compositions from biomass or biomass-containing compositions. Examples of dehydration include centrifugation, evaporation, heating, tangential flow filtration, and vacuum filtration. Further downstream treatment may follow dehydration.

[0066] Drying is understood as the process of removing water from biomass or a composition containing biomass to produce biomass that consists of all its components but is substantially free of water. Examples of drying include drum drying, belt drying, freeze drying, spray drying, and vacuum drying.

[0067] Purifying carbon dioxide from waste gases originating from production or combustion processes is understood to mean obtaining a volume consisting essentially of only carbon dioxide, wherein other elements of the waste gas are substantially removed by means of apparatus and methods known in the art (e.g., by using electrostatic precipitators or bag filters to remove ash and other particulate matter, by using denitrification units to remove nitrogen oxides, by using wet scrubbers, spray dry scrubbers, or dry adsorbent injection systems to remove sulfur oxides). Carbon dioxide can be captured during post-combustion processes by separation methods known in the art, for example, by using solvents such as amines to form carbonates. Carbon dioxide is absorbed by a solvent, and then released by heat to form a highly purified carbon dioxide stream.

[0068] Biologically available nitrogen is understood to refer to all types of nitrogen that are readily absorbed by microorganisms, including, for example, urea, ammonia, and amino acids. For clarity, it does not include molecular nitrogen (N2).

[0069] Process limiting is understood as the condition in which a substance can be measured at or near zero in the liquid phase.

[0070] A chemostat is understood to mean a bioreactor in which the chemical environment is maintained in a more or less stable state relative to, for example, microbial concentration, pH, (dissolved) gaseous substrate, nutrient composition, liquid volume, and other parameters known to those skilled in the art.

[0071] Invention Embodiments

[0072] This invention relates to a method for producing biomass containing at least 65% protein by dry weight using hydrogen-oxidizing bacteria grown in a bioreactor with an economically advantageous substrate conversion yield and high productivity, preferably using a high operating microbial concentration. To this end, relevant background process conditions and control parameters have been established for continuous fermentation using hydrogen-oxidizing bacteria. Appropriately controlling the availability of the growth substrate (gas, inorganic nitrogen, or phosphate) can improve the overall biomass productivity of the gas fermentation system while optimizing the protein concentration within the produced biomass, thereby producing optimal biomass.

[0073] There are various potential sources of gaseous carbon and energy that can be used for the growth of chemoautotrophic microorganisms used in human and animal nutrition products and other chemicals. These sources include, but are not limited to, industrial exhaust gases, industrial flue gases, and industrial byproducts, as well as direct air capture gases and in-situ electrochemical production gases. Chemoautotrophic metabolism, as used herein, refers to a metabolic pattern in which microorganisms take up inorganic carbon (e.g., by capturing carbon dioxide or formate or methanol as the primary carbon source) and obtain energy from chemical sources (e.g., by oxidizing hydrogen). By converting inorganic carbon into organic carbon, these microorganisms act as primary producers in the natural environment. Many of these chemoautotrophic microorganisms can be cultured in bioreactors with direct or indirect gaseous feedstocks for the commercial production of biomass, which can be processed into nutritional products such as animal feed, companion animal feed, or even human food.

[0074] Preferably, the microorganisms are fed with an indirect industrial waste gas feedstock that has been purified, filtered, and / or concentrated. In addition to the desired gaseous substrate for microbial growth, the industrial waste gas also contains other elements that can reduce the quality of microbial growth and / or the biomass produced.

[0075] Therefore, the method of the present invention is a method for producing biomass from hydrogen-oxidizing microorganisms using one or more input streams containing one or more gaseous substrates, said biomass comprising at least 65% protein of total biomass on a dry weight basis, said gaseous substrates comprising hydrogen and / or oxygen and / or carbon dioxide, said method comprising contacting microorganisms in a liquid phase with a nutrient composition comprising compounds containing carbon and / or nitrogen and / or phosphorus and the gaseous substrates, wherein the input streams and nutrient compositions are controlled and wherein biomass is produced at a rate greater than 10 g / L / day.

[0076] The biomass according to the present invention comprises cell clusters of microorganisms and / or their products, preferably, the biomass is a cell cluster of microorganisms and / or their products, more preferably, the biomass is a cell cluster of microorganisms.

[0077] According to the method of the present invention, most preferably, one or more gaseous substrates comprise hydrogen, oxygen, and carbon dioxide. Preferably, one or more gaseous substrates comprise hydrogen and oxygen. Preferably, one or more gaseous substrates comprise hydrogen and carbon dioxide. Preferably, one or more gaseous substrates comprise oxygen and carbon dioxide. Preferably, one or more gaseous substrates comprise hydrogen. Preferably, one or more gaseous substrates comprise oxygen. Preferably, one or more gaseous substrates comprise carbon dioxide.

[0078] According to the method of the present invention, most preferably, the nutrient composition comprises a carbon-containing compound, a nitrogen-containing compound, and a phosphorus-containing compound. Preferably, the nutrient composition comprises a carbon-containing compound and a nitrogen-containing compound. Preferably, the nutrient composition comprises a carbon-containing compound and a phosphorus-containing compound. Preferably, the nutrient composition comprises a nitrogen-containing compound. Preferably, the nutrient composition comprises a phosphorus-containing compound.

[0079] Therefore, one embodiment of the method according to the invention is a method for producing biomass from hydrogen-oxidizing microorganisms using one or more input streams containing one or more gaseous substrates, said biomass comprising at least 65% protein of total biomass on a dry weight basis, said gaseous substrates comprising hydrogen and oxygen, said method comprising contacting microorganisms in a liquid phase with a nutrient composition comprising nitrogen- and phosphorus-containing compounds and the gaseous substrates, wherein said one or more gaseous substrates comprise carbon dioxide and / or said nutrient composition comprises carbon-containing compounds, wherein the input streams and nutrient composition are controlled and wherein biomass is produced at a rate greater than 10 g / L / day.

[0080] Biomass produced by the method of the present invention is produced at a rate greater than 10 g / L / day. Preferably, the rate is greater than 10.5 g / L / day, 11.0 g / L / day, 11.5 g / L / day, 12.0 g / L / day, 12.5 g / L / day, 13.0 g / L / day, 13.5 g / L / day, 14.0 g / L / day, 14.5 g / L / day, 15.0 g / L / day, 15.5 g / L / day, 16.0 g / L / day, 16.5 g / L / day, 17.0 g / L / day, 17.5 g / L / day, 18.0 g / L / day, 18.5 g / L / day, 19.0 g / L / day, 19.5 g / L / day, or 20.0 g / L / day. Preferably, biomass is produced at the following rates: 10.0 g / L / day to 100 g / L / day, 10.0 g / L / day to 90 g / L / day, 10.0 g / L / day to 80 g / L / day, 10.0 g / L / day to 50 g / L / day, 10.5 g / L / day to 100 g / L / day, 10.5 g / L / day to 90 g / L / day, 10.5 g / L / day to 80 g / L / day, 10.5 g / L / day to 50 g / L / day, 11.0 g / L / day to 100 g / L / day, 11.0 g / L / day to 100 g / L / day. 1 / day to 90g / l / day, 11.0g / l / day to 80g / l / day, 11.0g / l / day to 50g / l / day, 11.5g / l / day to 100g / l / day, 11.5g / l / day to 90g / l / day, 11.5g / l / day to 80g / l / day, 11.5g / l / day to 50g / l / day, 12.0g / l / day to 100g / l / day, 12.0g / l / day to 90g / l / day, 12.0g / l / day to 80g / l / day, 12.0g / l / day to 50g / l / day 0 g / L / day, 12.5 g / L / day to 100 g / L / day, 12.5 g / L / day to 90 g / L / day, 12.5 g / L / day to 80 g / L / day, 12.5 g / L / day to 50 g / L / day, 13.0 g / L / day to 100 g / L / day, 13.0 g / L / day to 90 g / L / day, 13.0 g / L / day to 80 g / L / day, 13.5 g / L / day to 50 g / L / day, 13.5 g / L / day to 100 g / L / day, 13.5 g / L / day to 90 g / L / day, 13.5g / l / day to 80g / l / day, 13.5g / l / day to 50g / l / day, 14.0g / l / day to 100g / l / day, 14.0g / l / day to 90g / l / day, 14.0g / l / day to 80g / l / day, 14.0g / l / day to 50g / l / day, 14.5g / l / day to 100g / l / day, 14.5g / l / day to 90g / l / day, 14.5g / l / day to 80g / l / day, 14.5g / l / day to 50g / l / day, 15.0 g / l / day to 100 g / l / day, 15.0 g / l / day to 90 g / l / day, 15.0 g / l / day to 80 g / l / day, 15.0 g / l / day to 50 g / l / day, 15.5 g / l / day to 100 g / l / day, 15.5 g / l / day to 90 g / l / day, 15.5 g / l / day to 80 g / l / day, 15.5 g / l / day to 50 g / l / day, 16 0.0 g / L / day to 100 g / L / day, 16.0 g / L / day to 90 g / L / day, 16.0 g / L / day to 80 g / L / day, 16.0 g / L / day to 50 g / L / day, 16.5 g / L / day to 100 g / L / day, 16.5 g / L / day to 90 g / L / day, 16.5 g / L / day to 80 g / L / day, 16.5 g / L / day to 50 g / L / day, 17 0.0 g / l / day to 100 g / l / day, 17.0 g / l / day to 90 g / l / day, 17.0 g / l / day to 80 g / l / day, 17.0 g / l / day to 50 g / l / day, 18.0 g / l / day to 100 g / l / day, 18.0 g / l / day to 90 g / l / day, 18.0 g / l / day to 80 g / l / day, 18.0 g / l / day to 50 g / l / day, 1 9.0 g / L / day to 100 g / L / day, 19.0 g / L / day to 90 g / L / day, 19.0 g / L / day to 80 g / L / day, 19.0 g / L / day to 50 g / L / day, 20.0 g / L / day to 100 g / L / day, 20.0 g / L / day to 90 g / L / day, 20.0 g / L / day to 80 g / L / day, or 20.0 g / L / day to 50 g / L / day.

[0081] Furthermore, using a highly concentrated gaseous substrate source reduces the volume of other non-substrate gases in the microbial environment, thereby improving the efficiency of the added gaseous substrate and enabling improved control over the amount of gaseous substrate in the microbial environment. The gaseous substrate can be added to the liquid phase alone or as any premixed combination. According to the invention, it is preferable that the gaseous substrate is added with the lowest possible concentration of non-substrate gases such as nitrogen or carbon monoxide. Adding one or more gaseous substrates to the liquid phase includes contacting the gaseous substrate with the liquid phase, such that the gaseous substrate is mixed with at least a portion of the liquid phase. When one or more gaseous substrates are added to the liquid phase in a certain proportion, those skilled in the art will understand that one or more gaseous substrates can be added simultaneously or subsequently.

[0082] Preferably, the concentration of the concentrated gaseous substrate is: 2% to 100% (v / v), 5% to 100% (v / v), 10% to 100% (v / v), 20% to 100% (v / v), 30% to 100% (v / v), 40% to 100% (v / v), 50% to 100% (v / v), 60% to 100% (v / v), 70% to 100% (v / v), 80% to 100% (v / v), 90% to 100% (v / v), 95% to 100% (v / v), 98% to 100% (v / v), 99% to 100% (v / v), 2% to 90% (v / v), 5% to 90% (v / v), 5% to 90% (v / v), 90% to 10 ... 0% (v / v), 10% to 90% (v / v), 20% to 90% (v / v), 30% to 90% (v / v), 40% to 90% (v / v), 50% to 90% (v / v), 60% to 90% (v / v), 70% to 90% (v / v), 80% to 90% (v / v), 2% to 80% (v / v), 5% to 80% (v / v), 10% to 80% (v / v), 20% to 80% (v / v), 30% to 80% (v / v), 40% to 80% (v / v), 50% to 80% (v / v), 60% to 80% (v / v), 70% to 80% (v / v) 2% to 70% (v / v), 5% to 70% (v / v), 10% to 70% (v / v), 20% to 70% (v / v), 30% to 70% (v / v), 40% to 70% (v / v), 50% to 70% (v / v), 60% to 70% (v / v), 2% to 60% (v / v), 5% to 60% (v / v), 10% to 60% (v / v), 20% to 60% (v / v), 30% to 60% (v / v), 40% to 60% (v / v), 50% to 60% (v / v), 2% to 50% (v / v), 5% to 50% (v / v), 10% to 50% (v / v) ), 20% to 50% (v / v), 30% to 50% (v / v), 40% to 50% (v / v), 2% to 40% (v / v), 5% to 40% (v / v), 10% to 40% (v / v), 20% to 40% (v / v), 30% to 40% (v / v), 2% to 30% (v / v), 5% to 30% (v / v), 10% to 30% (v / v), 20% to 30% (v / v), 2% to 20% (v / v), 5% to 20% (v / v), 10% to 20% (v / v), 2% to 10% (v / v), 5% to 10% (v / v), or 2% to 5% (v / v).

[0083] Using highly concentrated gaseous substrate sources reduces the volume of other non-substrate gases in the microbial environment, thus ideally resulting in a gas phase consisting only of hydrogen, oxygen, and carbon dioxide.

[0084] Therefore, the present invention also provides a method wherein the gas phase consists essentially of only hydrogen, oxygen and carbon dioxide.

[0085] Preferably, the input stream is controlled by adding hydrogen gas at a concentration of 10% to 100% (v / v), oxygen gas at a concentration of 2% to 100% (v / v), and carbon dioxide gas at a concentration of 2% to 100% (v / v), either alone or as any premixed combination thereof, to the liquid phase. More preferably, the input stream is controlled by adding hydrogen gas at a concentration of 80% to 100% (v / v), oxygen gas at a concentration of 20% to 100% (v / v), and carbon dioxide gas at a concentration of 5% to 100% (v / v), either alone or as any premixed combination thereof, to the liquid phase. More preferably, the input flow is controlled by adding to the liquid phase, alone or as part of any premixed combination, at concentrations of 20% to 100% (v / v), 30% to 100% (v / v), 40% to 100% (v / v), 50% to 100% (v / v), 60% to 100% (v / v), 70% to 100% (v / v), 80% to 100% (v / v), 90% to 100% (v / v), 95% to 100% (v / v), 10% to 90% (v / v), 20% to Hydrogen gas at concentrations of 90% (v / v), 30% to 90% (v / v), 40% to 90% (v / v), 50% to 90% (v / v), 60% to 90% (v / v), 70% to 90% (v / v), 80% to 90% (v / v), 10% to 80% (v / v), 20% to 80% (v / v), 30% to 80% (v / v), 40% to 80% (v / v), 50% to 80% (v / v), 60% to 80% (v / v), or 70% to 80% (v / v).Concentrations of 2% to 100% (v / v), 5% to 100% (v / v), 10% to 100% (v / v), 20% to 100% (v / v), 30% to 100% (v / v), 40% to 100% (v / v), 50% to 100% (v / v), 60% to 100% (v / v), 70% to 100% (v / v), 80% to 100% (v / v), 90% to 100% (v / v), 95% to 100% (v / v), 2% to 90% (v / v), 5% to 90% (v / v), 10% to 90% (v / v), and 20% to 90% (v / v) ), 30% to 90% (v / v), 40% to 90% (v / v), 50% to 90% (v / v), 60% to 90% (v / v), 70% to 90% (v / v), 80% to 90% (v / v), 2% to 80% (v / v), 5% to 80% (v / v), 10% to 80% (v / v), 20% to 80% (v / v), 30% to 80% (v / v), 40% to 80% (v / v), 50% to 80% (v / v), 60% to 80% (v / v), 70% to 80% (v / v), 2% to 70% (v / v), 5% to 70% (v / v) ), 10% to 70% (v / v), 20% to 70% (v / v), 30% to 70% (v / v), 40% to 70% (v / v), 50% to 70% (v / v), 60% to 70% (v / v), 2% to 60% (v / v), 5% to 60% (v / v), 10% to 60% (v / v), 20% to 60% (v / v), 30% to 60% (v / v), 40% to 60% (v / v), 50% to 60% (v / v), 2% to 50% (v / v), 5% to 50% (v / v), 10% to 50% (v / v), 20% to 50% (v / v) ), 30% to 50% (v / v), 40% to 50% (v / v), 2% to 40% (v / v), 5% to 40% (v / v), 10% to 40% (v / v), 20% to 40% (v / v), 30% to 40% (v / v), 2% to 30% (v / v), 5% to 30% (v / v), 10% to 30% (v / v), 20% to 30% (v / v), 2% to 20% (v / v), 5% to 20% (v / v), 10% to 20% (v / v), 2% to 10% (v / v), 5% to 10% (v / v), or 2% to 5% (v / v) of oxygen,And concentrations of 2% to 100% (v / v), 5% to 100% (v / v), 10% to 100% (v / v), 20% to 100% (v / v), 30% to 100% (v / v), 40% to 100% (v / v), 50% to 100% (v / v), 60% to 100% (v / v), 70% to 100% (v / v), 80% to 100% (v / v), 90% to 100% (v / v), 95% to 100% (v / v), 2% to 90% (v / v), 5% to 90% (v / v), 10% to 90% (v / v), 20% to 90% (v / v) v), 30% to 90% (v / v), 40% to 90% (v / v), 50% to 90% (v / v), 60% to 90% (v / v), 70% to 90% (v / v), 80% to 90% (v / v), 2% to 80% (v / v), 5% to 80% (v / v), 10% to 80% (v / v), 20% to 80% (v / v), 30% to 80% (v / v), 40% to 80% (v / v), 50% to 80% (v / v), 60% to 80% (v / v), 70% to 80% (v / v), 2% to 70% (v / v), 5% to 70% (v / v) ), 10% to 70% (v / v), 20% to 70% (v / v), 30% to 70% (v / v), 40% to 70% (v / v), 50% to 70% (v / v), 60% to 70% (v / v), 2% to 60% (v / v), 5% to 60% (v / v), 10% to 60% (v / v), 20% to 60% (v / v), 30% to 60% (v / v), 40% to 60% (v / v), 50% to 60% (v / v), 2% to 50% (v / v), 5% to 50% (v / v), 10% to 50% (v / v), 20% to 50% (v / v) Carbon dioxide at concentrations of 30% to 50% (v / v), 40% to 50% (v / v), 2% to 40% (v / v), 5% to 40% (v / v), 10% to 40% (v / v), 20% to 40% (v / v), 30% to 40% (v / v), 2% to 30% (v / v), 5% to 30% (v / v), 10% to 30% (v / v), 20% to 30% (v / v), 2% to 20% (v / v), 5% to 20% (v / v), 10% to 20% (v / v), 2% to 10% (v / v), 5% to 10% (v / v), or 2% to 5% (v / v). Even more preferably, the input stream is controlled by adding hydrogen gas at a concentration of 70% to 100% (v / v), oxygen gas at a concentration of 20% to 100% (v / v), and carbon dioxide gas at a concentration of 5% to 100% (v / v), either alone or as any premixed combination thereof, to the liquid phase.

[0086] The substrate gas is preferably added with the lowest possible concentration of a non-substrate gas, such as nitrogen or carbon monoxide.

[0087] Advantageously, microorganisms are fed with indirect industrial waste gas feedstock that has been purified, filtered, and / or concentrated. Preferably, the gaseous substrate is derived from waste gas from a production or combustion process. More preferably, hydrogen is derived from waste gas from a production or combustion process. Even more preferably, carbon dioxide is derived from waste gas from a production or combustion process.

[0088] Preferably, the carbon dioxide from the waste gas from the production or combustion process is purified and concentrated to a concentration of 20% to 100% (v / v). More preferably, the carbon dioxide is purified and concentrated to the following concentrations: 30% to 100% (v / v), 40% to 100% (v / v), 50% to 100% (v / v), 60% to 100% (v / v), 70% to 100% (v / v), 80% to 100% (v / v), 90% to 100% (v / v), 95% to 100% (v / v), 99% to 100% (v / v), 20% to 90% (v / v), 30% to 90% (v / v), 40% to 90% (v / v), 50% to 90% (v / v), 60% to 90% (v / v), 70% to 90% (v / v), 80% to 90% (v / v), 20% to 80% (v / v), 30% to 80% (v / v). 0% (v / v), 40% to 80% (v / v), 50% to 80% (v / v), 60% to 80% (v / v), 70% to 80% (v / v), 20% to 70% (v / v), 30% to 70% (v / v), 40% to 70% (v / v), 50% to 70% (v / v), 60% to 70% (v / v), 20% to 60% (v / v), 30% to 60% (v / v), 40% to 60% (v / v), 50% to 60% (v / v), 20% to 50% (v / v), 30% to 50% (v / v), 40% to 50% (v / v), 20% to 40% (v / v), 30% to 40% (v / v), or 20% to 30% (v / v).

[0089] Chemoautotrophic metabolism is primarily found in many bacteria, including but not limited to purple non-sulfur bacteria such as *Rhodobacter capsulatus*, *Rhodobacter sphaeroides*, and *Rhodopsis damonaspalustris*, pseudomonads such as *Pseudomonas carboxydovorans*, aquagenic bacteria such as *Hydrogenobacter thermophilus*, methanogenic bacteria such as *Methanobacterium thermoautotrophicum*, alpha-proteobacteria such as *Xanthobacter flavus*, beta-proteobacteria such as *Ralstonia metallidurans* and *Hookworm copper-loving bacteria*, and gamma-proteobacteria such as *Hydrogenovibrio*. Microorganisms such as *Helicobacter pylori* (e.g., *Marinus*), ε-proteobacteria, acetic acid-producing bacteria such as *Acetobacterium woodii*, or other microorganisms expressing hydrogen uptake and carbon dioxide fixation metabolism, whether endogenous or introduced through genetic manipulation, mutation, selection, or directed evolution, can all perform heterotrophic and phototrophic metabolism, or a hybrid metabolism using both energy and carbon sources. They may use hydrogen as an energy source and carbon dioxide as a carbon source. Carbon monoxide can also serve as both an energy and carbon source.

[0090] Preferably, the microorganisms in the method according to the invention comprise bacteria selected from the following genera: *Rhodopseudomonas* sp., *Rhodospirillum* sp., *Rhodococcus* sp., *Rhodobacter* sp., *Rhizobium* sp., *Thiocapsasp.*, *Pseudomonas* sp., *Nocardia* sp., *Hydrogenomas* sp., *Hydrogenobacter* sp., *Hydrogenovibrio* sp., *Helicobacter* sp., *Xanthobacter* sp., *Hydrogenophaga* sp., *Bradyrhizobium* sp., and *Ralstonia* sp. The microorganisms include *Gordonium* sp., *Mycobacteria* sp., *Alcaligenes* sp., *Cupriavidus* sp., *Variovorax* sp., *Acidovorax* sp., *Anabaena* sp., *Scenedesmus* sp., *Chlamydomonas* sp., *Ankistrodesmus* sp., *Rhaphidium* sp., or *Arthrobacters* sp., and combinations thereof. More preferably, the microorganisms include bacteria selected from *Alcaligenes* or *Cupriavidus*. More preferably, the microorganisms include bacteria selected from *Alcaligenes*. More preferably, the microorganisms include bacteria selected from *Cupriavidus*. Even more preferably, the microorganisms include bacteria selected from the species *Cupriavidus hookworm*.

[0091] The properties of biomass produced by chemoautotrophic bacteria are directly related to its value in certain applications. For example, for animal feed and food applications, protein content and amino acid composition are crucial for nutritional quality.

[0092] The ratios of proteins, lipids, DNA, RNA, and other components in cellular biomass are mediated by growth conditions, growth rates, and carbon-to-nitrogen ratios. Therefore, control over substrate availability and general process conditions directly impacts the quantity and quality of biomass produced during fermentation. Maximum baseline productivity requires avoiding substrate limitations; however, as microbial density increases during fermentation, substrate utilization rates exceed supply, especially with gaseous substrates. This implies a need for high gas flow rates to establish and maximize productivity. However, achieving this is challenging in the case of explosive gas mixtures containing hydrogen and oxygen. Hydrogen-oxygen gas mixtures produce explosive mixtures when combined with a ratio exceeding 5% oxygen and 4% hydrogen, as disclosed, for example, in RK Kumar; FLAMMABILITY LIMITS OF HYDROGEN-OXYGEN-DILUENT MIXTURES; Journal of Fire Sciences, 1985.

[0093] In this method, oxygen and hydrogen inputs are controlled as part of a feedback loop to allow gases initially added to the system above their explosion limits, as they are initially injected into the liquid culture medium phase. The feedback control then ensures that the system fully utilizes oxygen and / or hydrogen, such that gases causing headspace are kept below their explosion safety limits, specifically 5% (v / v) for oxygen and 4% (v / v) for hydrogen under standard conditions. Furthermore, all three gas inputs are controlled such that oxygen or hydrogen remains the limiting gas within the system.

[0094] Combining these gas controls with defined parameters for background conditions, culture medium composition, inorganic nitrogen addition, and dilution rate advantageously enables the regulation of biomass composition through direct metabolic and physiological restrictions imposed on the microorganisms. Ammonium hydroxide and / or other bioavailable nitrogen sources are supplied to the process directly and / or by incorporation into the liquid culture medium input stream and / or the recirculated liquid stream, ensuring a minimum of 10 g atomic nitrogen per 100 g of biomass produced, controlled as part of a feedback loop in response to cell density and dilution rate in the liquid phase. In addition to the gaseous substrate, hydrogen-oxidizing bacteria require bioavailable nitrogen sources for protein production. This nitrogen is a major contributor to protein content, independent of molecular association in, for example, ammonium hydroxide (NH4OH) or ammonium chloride (NH3Cl). Since excess bioavailable nitrogen in the liquid phase is recycled, no maximum threshold is anticipated.

[0095] In order for microorganisms to produce sufficient protein, a sufficient amount of nitrogen (an essential component of amino acids) needs to be supplied. According to the method of the present invention, the controlled nutrient composition comprises adding at least 10 g of bioavailable nitrogen per 100 g of dry weight of biomass to be produced and present in the liquid phase. Preferably, the controlled nutrient composition comprises adding 10.0 g to 50,000 g of bioavailable nitrogen per 100 g of dry weight of biomass to be produced and present in the liquid phase.Preferably, the controlled nutrient composition includes adding 10.5g to 50,000g, 11.0g to 50,000g, 11.5g to 50,000g, 12.0g to 50,000g, 12.5g to 50,000g, 13.0g to 50,000g, 13.5g to 50,000g, 14.0g to 50,000g, 14.5g to 50,000g, 15.0g to 50,000g, 16g to 50,000g, 17g to 50,000g, 18g to 50,000g, 19g to 50,000g, or 20g to 50g of the dry weight of biomass to be produced and present in the liquid phase per 100g of biomass dry weight. 000g, 21g to 50000g, 22g to 50000g, 23g to 50000g, 24g to 50000g, 25g to 50000g, 26g to 50000g, 27g to 50000g, 28g to 50000g, 29g to 50000g, 30g to 50000g, 35g to 50000g, 40g to 50000g, 45g to 50000g, 50g to 50000g, 60g to 50000g, 70g to 50000g, 80g to 50000g, 90g to 50000g, 100g to 50000g, 110g 120g to 50000g, 130g to 50000g, 140g to 50000g, 150g to 50000g, 160g to 50000g, 170g to 50000g, 180g to 50000g, 190g to 50000g, 200g to 50000g, 250g to 50000g, 300g to 50000g, 350g to 50000g, 400g to 50000g, 450g to 50000g, 500g to 50000g, 1000g to 50000g, 1500g to 50000g, 20 00g to 50000g, 2500g to 50000g, 3000g to 50000g, 3500g to 50000g, 4000g to 50000g, 4500g to 50000g, 5000g to 50000g, 6000g to 50000g, 7000g to 50000g, 8000g to 50000g, 9000g to 50000g, 10000g to 50000g, 15000g to 50000g, 20000g to 50000g, 25000g to 50000g, or 30000g to 50000g of bioavailable nitrogen.

[0096] According to the present invention, controlling the nutrient composition preferably involves adding a suitable base, such as ammonium hydroxide (NH4OH) or NaOH, to the liquid phase to maintain the pH of the liquid phase at a physiologically suitable pH. Preferably, the physiologically suitable pH is 6.0 to 7.5 or 8.0, more preferably, the pH is 6.5 to 7.0.

[0097] According to the invention, controlling the nutrient composition preferably includes adding a growth medium with a pH of 1.0 to 3.0 or 4.0 before adding it to the liquid phase. Preferably, the pH of the growth medium is 2.2 to 3.2, or about 2.8. A low pH prevents precipitation of components of the growth medium during preparation. The components of the growth medium comprise those disclosed in Example 1 or components substantially similar to those disclosed in Example 1. The growth medium is prepared as disclosed in Example 1.

[0098] The liquid phase within the bioreactor is replaced with growth medium and / or other liquid input streams at a rate of 4% to 80% of its volume per hour to maximize the production rate, protein content, and quality of the biomass.

[0099] Certain specific replacement rates of the liquid phase within a bioreactor result in higher levels of protein content and quality in the biomass. Preferably, the liquid phase of the bioreactor used for growing and maintaining microorganisms is replaced per hour at the following volumes: 4% to 10%, 4% to 20%, 4% to 30%, 4% to 40%, 4% to 50%, 4% to 60%, 4% to 70%, 4% to 80%, 10% to 20%, 10% to 30%, 10% to 40%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 20% to 30%, 20% to 40%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 30% to 40%, 30% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 40% to 50%, 40% to 60%, 40% to 70%, 40% to 80%, 50% to 60%, 50% to 70%, 50% to 80%, 60% to 70%, 60% to 80%, or 70% to 80%. The consumption of gaseous substrate by microorganisms in the system is limited by the dissolution rate of the gas in the liquid phase and its distribution throughout the liquid phase. The volumetric mass transfer coefficient (kLa) represents the dissolution rate or efficiency of converting the concentration gradient of dissolved gas from its gas phase to its liquid phase. To obtain high utilization of gaseous substrate and subsequent high rates of biomass and protein production, it is necessary to maintain the highest possible kLa. Preferably, adding the gaseous substrate to the liquid phase at the highest possible concentration increases the driving force for gas dissolution and diffusion into the bulk liquid phase. Preferably, the gaseous substrate is added at its saturation concentration.

[0100] The gaseous substrate ratio, concentration, and microbial consumption need to be maintained through sufficient gas transfer to the liquid phase. Controlling the input flow includes maintaining gas transfer coefficients for hydrogen, oxygen, and carbon dioxide in the liquid phase at 1 / hour to 5000 / hour. Preferably, controlling the input flow includes maintaining gas transfer coefficients for hydrogen, oxygen, and carbon dioxide in the liquid phase at: 10 / hour to 5000 / hour, 20 / hour to 5000 / hour, 50 / hour to 5000 / hour, 100 / hour to 5000 / hour, 200 / hour to 5000 / hour, 400 / hour to 5000 / hour, 600 / hour to 5000 / hour, 800 / hour to 5000 / hour, 1000 / hour to 5000 / hour, 1500 / hour to 5000 / hour, 2000 / hour to 5000 / hour, 3000 / hour to 5000 / hour, 1 / hour to 3000 / hour. Hourly rates: 10 to 3000, 20 to 3000, 50 to 3000, 100 to 3000, 200 to 3000, 400 to 3000, 600 to 3000, 800 to 3000, 1000 to 3000, 1500 to 3000, 2000 to 3000, 1 to 2000, 10 to 2000, 20 to 2000, 50 to 2000, 100 Up to 2000 / hour, 200 / hour to 2000 / hour, 400 / hour to 2000 / hour, 600 / hour to 2000 / hour, 800 / hour to 2000 / hour, 1000 / hour to 2000 / hour, 1500 / hour to 2000 / hour, 1 / hour to 1500 / hour, 10 / hour to 1500 / hour, 200 / hour to 1500 / hour, 600 / hour to 1500 / hour, 800 / hour to 1500 / hour Hourly rates: 1000 to 1500, 1 to 1000, 10 to 1000, 20 to 1000, 50 to 1000, 100 to 1000, 200 to 1000, 400 to 1000, 600 to 1000, 800 to 1000, 1 to 800, 10 to 800, 20 to 800, 50 to 800, 100 to 800, 200 to 800.400 / hour to 800 / hour, 600 / hour to 800 / hour, 1 / hour to 600 / hour, 10 / hour to 600 / hour, 20 / hour to 600 / hour, 50 / hour to 600 / hour, 100 / hour to 600 / hour, 200 / hour to 600 / hour, 400 / hour to 600 / hour, 1 / hour to 400 / hour, 10 / hour to 400 / hour, 20 / hour to 400 / hour, 50 / hour to 400 / hour, 100 / hour to 400 / hour, 200 / hour to 400 / hour, 1 / hour to 200 / hour, 10 / hour to 200 / hour, 20 / hour to 200 / hour, 50 / hour to 200 / hour, 100 / hour to 200 / hour, 1 / hour to 100 / hour, 10 / hour to 100 / hour, 20 / hour to 100 / hour, 50 / hour to 100 / hour, 1 / hour to 50 / hour, 10 / hour to 50 / hour, 20 / hour to 50 / hour, 1 / hour to 20 / hour, 10 / hour to 20 / hour, or 1 / hour to 10 / hour.

[0101] Certain specific molar ratios of gaseous substrate input result in higher levels of protein content and quality in the biomass. Preferably, controlling the input stream involves adding hydrogen:oxygen:carbon dioxide in a molar ratio of 2 to 80:0.25 to 20:0.25 to 20 to the liquid phase.More preferably, controlling the input flow includes adding hydrogen:oxygen:carbon dioxide in the liquid phase at the following molar ratios: 3.88 to 51.94:0.85 to 2:0.75 to 2, 2 to 51.94:0.85 to 2:0.75 to 2, 3 to 51.94:0.85 to 2:0.75 to 2, 5 to 51.94:0.85 to 2:0.75 to 2, 6 to 51.94:0.85 to 2:0.75 to 2, 8 to 51.94:0.85 to 2:0.75 to 2, 10 to 51.94:0.85 to 2:0.75 to 2, 2 to 60:0.85 to 2:0.75 to 2, 3 to 60:0.85 to 2:0.75 2, 5 to 60: 0.85 to 2: 0.75 to 2, 6 to 60: 0.85 to 2: 0.75 to 2, 8 to 60: 0.85 to 2: 0.75 to 2, 10 to 60: 0.85 to 2: 0.75 to 2, 2 to 80: 0.85 to 2: 0.75 to 2, 3 to 80: 0.85 to 2: 0.75 to 2, 5 to 80: 0.85 to 2: 0.75 to 2, 6 to 80: 0.85 to 2: 0.75 to 2, 8 to 80: 0.85 to 2: 0.75 to 2, 10 to 80: 0.85 to 2: 0.75 to 2, 2 to 40: 0.85 to 2: 0.75 to 2, 3 to 40: 0.85 to 2: 0.75 2, 5 to 40: 0.85 to 2: 0.75 to 2, 6 to 40: 0.85 to 2: 0.75 to 2, 8 to 40: 0.85 to 2: 0.75 to 2, 10 to 40: 0.85 to 2: 0.75 to 2, 2 to 30: 0.85 to 2: 0.75 to 2, 3 to 30: 0.85 to 2: 0.75 to 2, 5 to 30: 0.85 to 2: 0.75 to 2, 6 to 30: 0.85 to 2: 0.75 to 2, 8 to 30: 0.85 to 2: 0.75 to 2, 10 to 30: 0.85 to 2: 0.75 to 2, 2 to 20: 0.85 to 2: 0.75 to 2, 3 to 20: 0.85 to 2: 0.75 2, 5 to 20: 0.85 to 2: 0.75 to 2, 6 to 20: 0.85 to 2: 0.75 to 2, 8 to 20: 0.85 to 2: 0.75 to 2, 10 to 20: 0.85 to 2: 0.75 to 2, 2 to 10: 0.85 to 2: 0.75 to 2, 3 to 10: 0.85 to 2: 0.75 to 2, 5 to 10: 0.85 to 2: 0.75 to 2, 6 to 10: 0.85 to 2: 0.75 to 2, 8 to 10: 0.85 to 2: 0.75 to 2, 2 to 6: 0.85 to 2: 0.75 to 2, 3 to 6: 0.85 to 2: 0.75 to 2, 5 to 6: 0.85 to 2: 0.75 to 2.

[0102] 3.88 to 51.94: 0.5 to 2: 0.75 to 2, 3.88 to 51.94: 0.25 to 2: 0.75 to 2, 3.88 to 51.94: 0.25 to 3: 0.75 to 2, 3.88 to 51.94: 0.5 to 3: 0.75 to 2, 3.88 to 51.94: 0.85 to 3: 0.75 to 2, 3.88 to 51.94: 0.25 to 4: 0.75 to 2, 3.8 8 to 51.94: 0.85 to 4: 0.75 to 2, 3.88 to 51.94: 2 to 4: 0.75 to 2, 3.88 to 51.94: 0.25 to 6: 0.75 to 2, 3.88 to 51.94: 0.5 to 6: 0.75 to 2, 3.88 to 51.94: 0.85 to 6: 0.75 to 2, 3.88 to 51.94: 2 to 6: 0.75 to 2, 3.88 to 51.94: 0.25 to 8: 0.75 to 2, 3.88 to 51.94: 0.5 to 8: 0.75 to 2, 3.88 to 51.94: 0.85 to 8: 0.75 to 2, 3.88 to 51.94: 2 to 8: 0.75 to 2, 3.88 to 51.94: 4 to 8: 0.75 to 2, 3.88 to 51.94: 0.25 to 12: 0.75 to 2, 3.88 to 51.94: 0.5 to 12: 0.75 to 2, 3.88 to 51.94: 0.85 to 12: 0.75 to 2, 3.88 to 51.94: 2 to 12: 0 0.75 to 2, 3.88 to 51.94: 4 to 12: 0.75 to 2, 3.88 to 51.94: 0.25 to 20: 0.75 to 2, 3.88 to 51.94: 0.5 to 20: 0.75 to 2, 3.88 to 51.94: 0.85 to 20: 0.75 to 2, 3.88 to 51.94: 2 to 20: 0.75 to 2, 3.88 to 51.94: 4 to 20: 0.75 to 2, 3.88 to 51.94: 10 to 20: 0.75 to 2

[0103] 3.88 to 51.94:0.85 to 2:0.25 to 2, 3.88 to 51.94:0.85 to 2:0.5 to 2, 3.88 to 51.94:0.85 to 2:0.25 to 2.5, 3.88 to 51.94:0.85 to 2:0.5 to 2.5, 3.88 to 51.94:0.85 to 2:0.75 to 2.5, 3.88 to 51.94:0.85 to 2:0.25 to 3, 3.88 to 51.94:0.85 to 2:0.5 to 3, 3.88 to 51.94:0.85 to 2:0.75 to 3, 3.88 to 51.94:0.85 to 2:1 to 3, 3.88 to 51.94:0.85 to 2:1.5 to 3, 3.88 to 51.94:0.85 to 2:2 to 3, 3.88 to 51.94:0.85 to 2:0.25 to 5, 3.88 to 51.94:0.85 to 2:0.5 to 5, 3.88 to 51.94:0.85 to 2:0.75 to 5, 3.88 to 51.94:0.85 to 2:1 to 5, 3.88 to 51.94:0.85 to 2:1.5 to 5, 3.88 to 51.94:0.85 to 2:2 to 5, 3.88 to 51.94:0.85 to 2:0.25 to 8, 3.88 to 51.94:0.85 to 2:0.5 to 8, 3.88 to 51.94:0.85 to 2:0.7 5 to 8, 3.88 to 51.94:0.85 to 2:1 to 8, 3.88 to 51.94:0.85 to 2:1.5 to 8, 3.88 to 51.94:0.85 to 2:2 to 8, 3.88 to 51.94:0.85 to 2:4 to 8, 3.88 to 51.94:0.85 to 2:0.25 to 12, 3.88 to 51.94:0.85 to 2:0.5 to 12, 3.88 to 51.94:0.85 to 2:0.75 to 12, 3.88 to 51.94:0.85 to 2:1 to 12, 3.88 to 51.94:0.85 to 2:1.5 to 12, 3.88 to 51.94:0.85 to 2:2 to 12 3.88 to 51.94:0.85 to 2:4 to 12, 3.88 to 51.94:0.85 to 2:8 to 12, 3.88 to 51.94:0.85 to 2:0.25 to 20, 3.88 to 51.94:0.85 to 2:0.5 to 20, 3.88 to 51.94:0.85 to 2:0.75 to 20, 3.88 to 51.94:0.85 to 2:1 to 20, 3.88 to 51.94:0.85 to 2:1.5 to 20, 3.88 to 51.94:0.85 to 2:2 to 20, 3.88 to 51.94:0.85 to 2:4 to 20, or 3.88 to 51.94:0.85 to 2:8 to 20.

[0104] Carbon, preferably carbon dioxide, is preferably not metabolically restricted, and this can be controlled using a range of possible methods, including, for example:

[0105] - Use a dissolved carbon dioxide probe to monitor dissolved carbon dioxide and use the obtained data as part of a feedback loop to maintain a concentration of approximately 1 mmol / L or higher by controlling the rate of addition of the input gas; or

[0106] - Maintain the ratio of hydrogen to carbon dioxide to less than 6 and the ratio of oxygen to carbon dioxide to less than 1.75 at the gas input point.

[0107] Regarding the addition of a specific molar ratio of gaseous substrate, it has been found that, according to the present invention, controlling the input flow preferably includes adding a hydrogen:oxygen mixture in the liquid phase at a molar ratio of 0.5:1 to 12:1. More preferably, controlling the input flow includes adding hydrogen:oxygen mixtures in the liquid phase at the following molar ratios: 0.5:1 to 10:1, 0.5:1 to 8:1, 0.5:1 to 6:1, 0.5:1 to 4:1, 0.5:1 to 2:1, 1:1 to 12:1, 1:1 to 10:1, 1:1 to 9:1, 1:1 to 8:1, 1:1 to 7:1, 1:1 to 6:1, 1:1 to 5:1, 1:1 to 4:1, 1:1 to 3:1, 1:1 to 2:1, 2:1 to 12:1, 2:1 to 10:1, 2:1 to 8:1, 2:1 to 6:1. 2:1 to 4:1, 2.5:1 to 12:1, 2.5:1 to 10:1, 2.5:1 to 8:1, 2.5:1 to 6:1, 2.5:1 to 5:1, 2.5:1 to 4:1, 3:1 to 12:1, 3:1 to 10:1, 3:1 to 8:1, 3:1 to 6:1, 3:1 to 4:1, 4:1 to 12:1, 4:1 to 10:1, 4:1 to 8:1, 4:1 to 6:1, 5:1 to 12:1, 5:1 to 10:1, 5:1 to 8:1, 6:1 to 12:1, 6:1 to 10:1, or 6:1 to 8:1. Even more preferably, controlling the input flow includes adding hydrogen to oxygen in the liquid phase at a molar ratio of 1:1 to 10:1, 1:1 to 9:1, 1:1 to 8:1, 1:1 to 7:1, 1:1 to 6:1, or 1:1 to 5:1. Most preferably, controlling the input flow includes adding hydrogen to oxygen in the liquid phase at a molar ratio of 1.5 or 1.7:1 to 10:1, 1.5 or 1.7:1 to 9:1, 1.5 or 1.7:1 to 8:1, 1.5 or 1.7:1 to 7:1, 1.5 or 1.7:1 to 6 or 6.6:1, or 1.5 or 1.7:1 to 5:1.

[0108] The term "gas retention" is defined as the volume fraction of gas (including input gas and any other gas formed in the liquid phase) in a bioreactor. In partially or completely closed bioreactor systems, and in bioreactor systems employing gas-phase and / or gas-liquid phase recirculation, the gas retention composition ratio is preferably maintained by matching the gas input ratio to the gas usage ratio. In open systems, the desired gas retention composition is preferably controlled by using this composition or a very similar composition as the average input gas composition. Thus, regarding maintaining a certain molar ratio of gaseous substrate, it has been found that controlling the input flow according to the invention preferably involves maintaining a hydrogen:oxygen gas retention molar ratio in the liquid phase of 0.5:1 to 7:1. More preferably, controlling the input flow includes maintaining the molar ratio of hydrogen to oxygen gas retention in the liquid phase at 0.5:1 to 7:1, 0.5:1 to 6:1, 0.5:1 to 4:1, 0.5:1 to 2:1, 1:1 to 7:1, 1:1 to 6:1, 1:1 to 5:1, 1:1 to 4:1, 1:1 to 3:1, 1:1 to 2:1, 2:1 to 6:1, 2:1 to 4:1, 2:1 to 3 or 3.5:1, 2.5:1 to 6:1, 2.5:1 to 5:1, 2.5:1 to 4:1, 3:1 to 6:1, 3:1 to 4:1, 4:1 to 7:1, or 4:1 to 6:1. Even more preferably, controlling the input flow includes maintaining the molar ratio of hydrogen to oxygen gas retention in the liquid phase at 1.2:1 to 5:1, 1.2:1 to 4.5:1, 1.2:1 to 4:1, 1.2:1 to 3.5:1, 1.2:1 to 3:1, or 1.2:1 to 2.5:1. Most preferably, controlling the input flow includes maintaining the molar ratio of hydrogen to oxygen gas retention in the liquid phase at 1.5:1 to 2.5:1, 1.5:1 to 3:1, 1, or 1.5:1 to 3.5 or 4:1.

[0109] According to the present invention, in order to obtain biomass with high protein content at a high production rate, it has been found that oxygen, as a gaseous substrate, is preferably added to the liquid phase at the highest possible concentration, while also considering other parameters such as the hydrogen-to-oxygen ratio. Preferably, according to the present invention, a certain molar ratio of hydrogen to oxygen is added to the liquid phase: oxygen comprises adding oxygen to the liquid phase at a concentration of 5% to 100% (v / v). More preferably, according to the invention, hydrogen is added to the liquid phase in a certain molar ratio: oxygen includes adding oxygen to the liquid phase at a concentration of 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% to 100% (v / v), or 5% to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% (v / v). Even more preferably, according to the invention, hydrogen is added to the liquid phase in a certain molar ratio: oxygen includes adding oxygen to the liquid phase at a concentration of 10% to 100% (v / v). Even more preferably, according to the invention, hydrogen is added to the liquid phase in a certain molar ratio: oxygen includes adding oxygen to the liquid phase at a concentration of 20% to 100% (v / v).

[0110] Because hydrogen, oxygen, and carbon dioxide have different solubilities in water, maintaining the molar ratio of dissolved hydrogen, oxygen, and carbon dioxide is important for optimal biomass production. Preferably, controlling the input flow includes maintaining the molar ratio of dissolved hydrogen:oxygen:carbon dioxide in the liquid phase at 1 to 60:0.5 to 20:0.5 to 20.More preferably, controlling the input flow includes maintaining the molar ratio of dissolved hydrogen:oxygen:carbon dioxide in the liquid phase at 3.183 to 12.748:0.795 to 4.25:0.75 to 2.0, 1 to 12.748:0.795 to 4.25:0.75 to 2.0, 2 to 12.748:0.795 to 4.25:0.75 to 2.0, 6 to 12.748:0.795 to 4.25:0.75 to 2.0, 1 to 20:0.795 to 4.25:0.75 to 2.0, 2 to 20:0.795 to 4.25:0.75 to 2.0, 3 to 20:0.795 to 4.25:0.75 to 2.0, and 6 to 20:0.7 95 to 4.25: 0.75 to 2.0, 1 to 30: 0.795 to 4.25: 0.75 to 2.0, 2 to 30: 0.795 to 4.25: 0.75 to 2.0, 3 to 30: 0.795 to 4.25: 0.75 to 2.0, 6 to 30: 0.795 to 4.25: 0.75 to 2.0, 12 to 30: 0.795 to 4.25: 0.75 to 2.0, 1 to 40: 0.795 to 4.25: 0.75 to 2.0, 2 to 40: 0.795 to 4.25: 0.75 to 2.0, 3 to 40: 0.795 to 4.25: 0.75 to 2.0, 6 to 40: 0.795 to 4.25: 0.75 2.0, 12 to 40: 0.795 to 4.25: 0.75 to 2.0, 20 to 40: 0.795 to 4.25: 0.75 to 2.0, 1 to 60: 0.795 to 4.25: 0.75 to 2.0, 2 to 60: 0.795 to 4.25: 0.75 to 2.0, 3 to 60: 0.795 to 4.25: 0.75 to 2.0, 6 to 60: 0.795 to 4.25: 0.75 to 2.0, 12 to 60: 0.795 to 4.25: 0.75 to 2.0, 20 to 60: 0.795 to 4.25: 0.75 to 2.0, 30 to 60: 0.795 to 4.25: 0.75 to 2.0, 1 to 8 :0.795 to 4.25:0.75 to 2.0, 2 to 8:0.795 to 4.25:0.75 to 2.0, 4 to 8:0.795 to 4.25:0.75 to 2.0, 6 to 8:0.795 to 4.25:0.75 to 2.0, 1 to 6:0.795 to 4.25:0.75 to 2.0, 2 to 6:0.795 to 4.25:0.75 to 2.0, 4 to 6:0.795 to 4.25:0.75 to 2.0, 1 to 4:0.795 to 4.25:0.75 to 2.0, 2 to 4:0.795 to 4.25:0.75 to 2.0, 1 to 2:0.795 to 4.25:0.75 to 2.0.

[0111] 3.183 to 12.748: 0.5 to 4.25: 0.75 to 2.0, 3.183 to 12.748: 1.5 to 4.25: 0.75 to 2.0, 3.183 to 12.748: 2.5 to 4.25: 0.75 to 2.0, 3.183 to 12.748: 0.5 to 8: 0.75 to 2.0, 3.183 to 12.748: 0.795 to 8:0. 75 to 2.0, 3.183 to 12.748: 1.5 to 8: 0.75 to 2.0, 3.183 to 12.748: 2.5 to 8: 0.75 to 2.0, 3.183 to 12.748: 4 to 8: 0.75 to 2.0, 3.183 to 12.748: 0.5 to 12: 0.75 to 2.0, 3.183 to 12.748: 0.795 to 12: 0.75 To 2.0, 3.183 to 12.748: 1.5 to 12: 0.75 to 2.0, 3.183 to 12.748: 2.5 to 12: 0.75 to 2.0, 3.183 to 12.748: 4 to 12: 0.75 to 2.0, 3.183 to 12.748: 8 to 12: 0.75 to 2.0, 3.183 to 12.748: 0.5 to 20: 0.75 to 2. 0, 3.183 to 12.748: 0.795 to 20: 0.75 to 2.0, 3.183 to 12.748: 1.5 to 20: 0.75 to 2.0, 3.183 to 12.748: 2.5 to 20: 0.75 to 2.0, 3.183 to 12.748: 4 to 20: 0.75 to 2.0, 3.183 to 12.748: 8 to 20: 0.75 to 2.0,

[0112] 3.183 to 12.748: 0.795 to 4.25: 0.25 to 2.0, 3.183 to 12.748: 0.795 to 4.25: 0.5 to 2.0, 3.183 to 12.748: 0.795 to 4.25: 1.25 to 2.0, 3.183 to 12.748: 0.795 to 4.25: 0.2 5 to 2.5, 3.183 to 12.748: 0.795 to 4.25: 0.5 to 2.5, 3.183 to 12.748: 0.795 to 4.25: 0.75 to 2.5, 3.183 to 12.748: 0.795 to 4.25: 1.25 to 2.5, 3.183 to 12.748: 0.795 to 4. 25: 0.25 to 3, 3.183 to 12.748: 0.795 to 4.25: 0.5 to 3, 3.183 to 12.748: 0.795 to 4.25: 0.75 to 3, 3.183 to 12.748: 0.795 to 4.25: 1.25 to 3, 3.183 to 12.748: 0.795 to 4.25 :2 to 3, 3.183 to 12.748: 0.795 to 4.25: 0.25 to 3, 3.183 to 12.748: 0.795 to 4.25: 0.5 to 5, 3.183 to 12.748: 0.795 to 4.25: 0.75 to 5, 3.183 to 12.748: 0.795 to 4.25: 1.25 To 5, 3.183 to 12.748: 0.795 to 4.25: 2 to 5, 3.183 to 12.748: 0.795 to 4.25: 3 to 5, 3.183 to 12.748: 0.795 to 4.25: 0.5 to 8, 3.183 to 12.748: 0.795 to 4.25: 0.75 to 8, 3.183 To 12.748:0.795 to 4.25:1.25 to 8, 3.183 to 12.748:0.795 to 4.25:2 to 8, 3.183 to 12.748:0.795 to 4.25:3 to 8, 3.183 to 12.748:0.795 to 4.25:5 to 8, 3.183 to 12.748:0. 795 to 4.25: 0.5 to 10, 3.183 to 12.748: 0.795 to 4.25: 0.75 to 10, 3.183 to 12.748: 0.795 to 4.25: 1.25 to 10, 3.183 to 12.748: 0.795 to 4.25: 2 to 10, 3.183 to 12.748: 0.7 95 to 4.25: 3 to 10, 3.183 to 12.748: 0.795 to 4.25: 5 to 10, 3.183 to 12.748: 0.795 to 4.25: 0.5 to 15, 3.183 to 12.748: 0.795 to 4.25: 0.75 to 15, 3.183 to 12.748: 0.795 to 4.25:1.25 to 15, 3.183 to 12.748:0.795 to 4.25:2 to 15, 3.183 to 12.748:0.795 to 4.25:3 to 15, 3.183 to 12.748:0.795 to 4.25:5 to 15, 3.183 to 12.748:0.795 to 4.25:10 to 15, 3.183 to 12.748:0.795 to 4.25:0.5 to 20, 3.183 to 12.74 8:0.795 to 4.25:0.75 to 20, 3.183 to 12.748:0.795 to 4.25:1.25 to 20, 3.183 to 12.748:0.795 to 4.25:2 to 20, 3.183 to 12.748:0.795 to 4.25:3 to 20, 3.183 to 12.748:0.795 to 4.25:5 to 20, or 3.183 to 12.748:0.795 to 4.25:10 to 20.

[0113] Accordingly, controlling the input flow includes maintaining the hydrogen concentration in the liquid phase at 0.5 mg / L to 20 mg / L, the oxygen concentration at 0.5 mg / L to 80 mg / L, and the carbon dioxide concentration at 20 mg / L to 2000 mg / L at a temperature of 28°C to 45°C and a gas phase pressure of 100 kPa to 2000 kPa. Preferably, controlling the input flow includes maintaining the concentrations of hydrogen, oxygen, and carbon dioxide at 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L, and 20 mg / L to 2000 mg / L, respectively; 2 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L, and 20 mg / L to 2000 mg / L; 5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L, and 20 mg / L to 2000 mg / L; 10 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L, and 20 mg / L to 2000 mg / L. 1 to 2000 mg / L; 0.5 mg / L to 15 mg / L, 0.5 mg / L to 10 mg / L and 50 mg / L to 250 mg / L; 0.5 mg / L to 15 mg / L, 0.5 mg / L to 80 mg / L and 20 mg / L to 2000 mg / L; 2 mg / L to 15 mg / L, 0.5 mg / L to 80 mg / L and 20 mg / L to 2000 mg / L; 5 mg / L to 15 mg / L, 0.5 mg / L to 80 mg / L and 20 mg / L to 2000 mg / L; 10 mg / L to 15 mg / L, 0.5 mg / L to 80 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 10 mg / L, 0.5 mg / L to 80 mg / L and 20 mg / L to 2000 mg / L; 2 mg / L to 10 mg / L, 0.5 mg / L to 80 mg / L and 20 mg / L to 2000 mg / L; 5 mg / L to 10 mg / L, 0.5 mg / L to 80 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 5 mg / L, 0.5 mg / L to 80 mg / L g / l and 20 mg / l to 2000 mg / l; 1.0 mg / l to 5 mg / l, 0.5 mg / l to 80 mg / l and 20 mg / l to 2000 mg / l; 2.0 mg / l to 5 mg / l, 0.5 mg / l to 80 mg / l and 20 mg / l to 2000 mg / l; 0.5 mg / l to 3 mg / l, 0.5 mg / l to 80 mg / l and 20 mg / l to 2000 mg / l; 1 mg / l to 3 mg / l, 0.5 mg / l to 80 mg / l and 20 mg / l to 2000 mg / l;

[0114] 0.5 mg / L to 20 mg / L, 2 mg / L to 80 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 5 mg / L to 80 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 10 mg / L to 80 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 20 mg / L to 80 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 40 mg / L to 80 mg / L and 20 mg / L 1 to 2000 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 50 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 2 mg / L to 50 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 5 mg / L to 50 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 10 mg / L to 50 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 20 mg / L to 50 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 20 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 2 mg / L to 20 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 5 mg / L to 20 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 10 mg / L to 20 mg / L and 20 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L mg / l, 0.5 mg / l to 10 mg / l and 20 mg / l to 2000 mg / l; 0.5 mg / l to 20 mg / l, 2 mg / l to 10 mg / l and 20 mg / l to 2000 mg / l; 0.5 mg / l to 20 mg / l, 5 mg / l to 10 mg / l and 20 mg / l to 2000 mg / l; 0.5 mg / l to 20 mg / l, 0.5 mg / l to 5 mg / l and 20 mg / l to 2000 mg / l; 0.5 mg / l to 20 mg / l, 2 mg / l to 5 mg / l and 20 mg / l to 2000 mg / l;

[0115] 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 50 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 100 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 200 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 500 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 1000 mg / L to 2000 mg / L; 0.5 mg / L to 20 mg / L 0.5 mg / L to 80 mg / L and 20 mg / L to 1500 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 50 mg / L to 1500 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 100 mg / L to 1500 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 200 mg / L to 1500 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 500 mg / L to 1500 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L 1000 mg / L to 1500 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 20 mg / L to 1000 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 50 mg / L to 1000 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 100 mg / L to 1000 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 200 mg / L to 1000 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 500 mg / L to 1000 mg / L mg / l; 0.5 mg / l to 20 mg / l, 0.5 mg / l to 80 mg / l and 20 mg / l to 500 mg / l; 0.5 mg / l to 20 mg / l, 0.5 mg / l to 80 mg / l and 50 mg / l to 500 mg / l; 0.5 mg / l to 20 mg / l, 0.5 mg / l to 80 mg / l and 100 mg / l to 500 mg / l; 0.5 mg / l to 20 mg / l, 0.5 mg / l to 80 mg / l and 200 mg / l to 500 mg / l; 0.5 mg / l to 20 mg / l, 0.5 mg / l to 80 mg / l and 20 mg / l to 250 mg / l; 0.5 mg / l to 20 mg / l, 0.5 mg / L to 80 mg / L and 50 mg / L to 250 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 100 mg / L to 250 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 20 mg / L to 100 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 50 mg / L to 100 mg / L; 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 20 mg / L to 50 mg / L; or 0.5 mg / L to 20 mg / L, 0.5 mg / L to 80 mg / L and 30 mg / L to 50 mg / L.

[0116] The concentration of dissolved gases in a liquid can preferably be measured by collecting a liquid sample under vacuum and maintaining it under vacuum until analysis by gas chromatography. Alternative, less accurate, and less preferred measurement methods are known in the art and include in-line measurements using, for example, fiber optic probes, impedance probes, heat transfer probes, or ultrasonic probes.

[0117] It has been found that maintaining a minimum hydrogen and / or oxygen transfer rate in the liquid phase is necessary for producing biomass containing at least 65% protein at a rate of at least 10 g / L / day, and further preferably maintaining a microbial concentration of at least 10 g / L in the liquid phase of the bioreactor. Theoretically, a maximum transfer rate is not required considering only biomass production rate and quality; however, commercial, cost, and safety considerations necessitate the application of a maximum transfer rate.

[0118] Preferably, according to the invention, controlling the input flow includes maintaining the hydrogen transfer rate in the liquid phase at at least 0.02 mol / L / hour and / or the oxygen transfer rate in the liquid phase at at least 0.003 mol / L / hour. More preferably, controlling the input flow includes maintaining the hydrogen transfer rate in the liquid phase at at least 0.03 mol / L / hour, 0.04 mol / L / hour, 0.05 mol / L / hour, 0.06 mol / L / hour, 0.07 mol / L / hour, 0.08 mol / L / hour, 0.09 mol / L / hour, 0.1 mol / L / hour, 0.11 mol / L / hour, 0.12 mol / L / hour, or 0.13 mol / L / hour. 0.14 mol / L / hour, 0.15 mol / L / hour, 0.16 mol / L / hour, 0.17 mol / L / hour, 0.18 mol / L / hour, 0.19 mol / L / hour, 0.2 mol / L / hour, 0.21 mol / L / hour, 0.22 mol / L / hour, 0.23 mol / L / hour, 0.24 mol / L / hour, 0.25 mol / L / hour, 0.3 mol / L / hour, 0.35 mol / L / hour, 0.4 mol / L / hour or 0.5 mol / L / hour, and / or the rate of oxygen transfer in the liquid phase is at least 0.005 mol / L / hour, 0.01 mol / L / hour, 0.015 mol / L / hour, 0.02 mol / L / hour, 0.025 mol / L / hour, 0.03 mol / L / hour, 0.035 mol / L / hour, 0.04 mol / L / hour, 0 0.045 mol / L / hour, 0.05 mol / L / hour, 0.06 mol / L / hour, 0.07 mol / L / hour, 0.08 mol / L / hour, 0.09 mol / L / hour, 0.1 mol / L / hour, 0.11 mol / L / hour, 0.12 mol / L / hour, 0.13 mol / L / hour, 0.14 mol / L / hour, 0.15 mol / L / hour, or 0.2 mol / L / hour.Even more preferably, controlling the input flow includes maintaining the hydrogen transfer rate in the liquid phase at at least 0.1 mol / L / h, 0.11 mol / L / h, 0.12 mol / L / h, 0.13 mol / L / h, 0.14 mol / L / h, 0.15 mol / L / h, 0.16 mol / L / h, 0.17 mol / L / h, 0.18 mol / L / h, 0.19 mol / L / h, 0.2 mol / L / h, 0.21 mol / L / h, 0.22 mol / L / h, 0.23 mol / L / h, 0.24 mol / L / h, 0.25 mol / L / h, or 0.3 mol / L / h and / or Or the rate of oxygen transfer in the liquid phase is at least 0.02 mol / L / hour, 0.025 mol / L / hour, 0.03 mol / L / hour, 0.035 mol / L / hour, 0.04 mol / L / hour, 0.045 mol / L / hour, 0.05 mol / L / hour, 0.06 mol / L / hour, 0.07 mol / L / hour, 0.08 mol / L / hour, 0.09 mol / L / hour, 0.1 mol / L / hour, 0.11 mol / L / hour, 0.12 mol / L / hour, 0.13 mol / L / hour, 0.14 mol / L / hour, 0.15 mol / L / hour, or 0.2 mol / L / hour.

[0119] Preferably, according to the present invention, controlling the input flow includes maintaining the hydrogen transfer rate in the liquid phase at 0.02 mol / L / hour to 3.0 mol / L / hour and / or the oxygen transfer rate in the liquid phase at 0.01 mol / L / hour to 0.4 mol / L / hour. More preferably, controlling the input flow includes maintaining the hydrogen transfer rate in the liquid phase at 0.04 mol / L / hour, 0.05 mol / L / hour, 0.06 mol / L / hour, 0.07 mol / L / hour, 0.08 mol / L / hour, 0.09 mol / L / hour, 0.1 mol / L / hour, 0.11 mol / L / hour, 0.12 mol / L / hour, 0.13 mol / L / hour, 0.14 mol / L / hour, 0.15 mol / L / hour, 0.16 mol / L / hour, and 0.17 mol / L / hour. 0.18 mol / L / hour, 0.19 mol / L / hour, 0.2 mol / L / hour, 0.21 mol / L / hour, 0.22 mol / L / hour, 0.23 mol / L / hour, 0.24 mol / L / hour, 0.25 mol / L / hour or 0.3 mol / L / hour to 0.35 mol / L / hour, 0.4 mol / L / hour, 0.45 mol / L / hour, 0.5 mol / L / hour, 0.55 mol / L / hour, 0.6 mol / L / hour, 0.8 mol / L / hour, The rates of oxygen transfer in the liquid phase are 0.9 mol / L / h, 1.0 mol / L / h, 1.1 mol / L / h, 1.2 mol / L / h, 1.3 mol / L / h, 1.4 mol / L / h, 1.5 mol / L / h, 2.0 mol / L / h, or 2.5 mol / L / h and / or 0.015 mol / L / h, 0.02 mol / L / h, 0.025 mol / L / h, 0.03 mol / L / h, 0.035 mol / L / h, 0.04 mol / L / h, and 0. 0.45 mol / L / hour, 0.05 mol / L / hour, 0.06 mol / L / hour, 0.07 mol / L / hour, 0.08 mol / L / hour, 0.09 mol / L / hour, 0.1 mol / L / hour, 0.11 mol / L / hour, 0.12 mol / L / hour, 0.13 mol / L / hour, 0.14 mol / L / hour, 0.15 mol / L / hour or 0.2 mol / L / hour to 0.25 mol / L / hour, 0.3 mol / L / hour or 0.35 mol / L / hour.Even more preferably, controlling the input flow includes maintaining the hydrogen transfer rate in the liquid phase at 0.1 mol / L / h, 0.11 mol / L / h, 0.12 mol / L / h, 0.13 mol / L / h, 0.14 mol / L / h, 0.15 mol / L / h, 0.16 mol / L / h, 0.17 mol / L / h, 0.18 mol / L / h, 0.19 mol / L / h, 0.2 mol / L / h, 0.21 mol / L / h, 0.22 mol / L / h, 0.23 mol / L / h, 0.24 mol / L / h, 0.25 mol / L / h, or 0.3 mol / L / h to 0.35 mol / L / h, 0.4 mol / L / h, 0.45 mol / L / h, 0.5 mol / L / h. The oxygen transfer rates in the liquid phase are 0.03 mol / L / hour, 0.035 mol / L / hour, 0.04 mol / L / hour, 0.045 mol / L / hour, 0.05 mol / L / hour, 0.06 mol / L / hour, 0.07 mol / L / hour or 0.08 mol / L / hour to 0.09 mol / L / hour, 0.1 mol / L / hour, 0.11 mol / L / hour, 0.12 mol / L / hour, 0.13 mol / L / hour, 0.14 mol / L / hour, 0.15 mol / L / hour, 0.2 mol / L / hour, 0.25 mol / L / hour or 0.3 mol / L / hour. Furthermore, more preferably, the oxygen transfer rate in the liquid phase is maintained at 0.04 mol / L / hour, 0.045 mol / L / hour, 0.05 mol / L / hour, 0.06 mol / L / hour, 0.07 mol / L / hour, 0.08 mol / L / hour, 0.09 mol / L / hour, 0.1 mol / L / hour to 0.11 mol / L / hour, 0.12 mol / L / hour, 0.13 mol / L / hour, 0.14 mol / L / hour, 0.15 mol / L / hour, 0.16 mol / L / hour, 0.18 mol / L / hour, or 0.2 mol / L / hour.

[0120] Preferably, the microorganisms utilize hydrogen, oxygen, and carbon dioxide at rates of 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h, and 0.01 mol / g / h to 0.5 mol / g / h, respectively. More preferably, the microorganisms utilize hydrogen, oxygen, and carbon dioxide at rates of: 0.05 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h, and 0.01 mol / g / h to 0.5 mol / g / h; 0.1 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h, and 0.01 mol / g / h to 0.5 mol / g / h; and 0.2 mol / g / h to 1.0 mol / g / h. 0.01 mol / g / hour, 0.01 mol / g / hour to 0.5 mol / g / hour, and 0.01 mol / g / hour to 0.5 mol / g / hour; 0.5 mol / g / hour to 1.0 mol / g / hour, 0.01 mol / g / hour to 0.5 mol / g / hour, and 0.01 mol / g / hour to 0.5 mol / g / hour; 0.03 mol / g / hour to 0.5 mol / g / hour, 0.01 mol / g / hour to 0.5 mol / g / hour, and 0.01 mol / g / hour to 0.5 mol / g / hour. Up to 0.5 mol / g / h; 0.05 mol / g / h to 0.5 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.1 mol / g / h to 0.5 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.2 mol / g / h to 0.5 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h; ...01 mol / g / h to 0.5 mol / g / h; 0.01 mol / g / h to 0.5 mol / g / h; 0.01 mol / g / h to 0.5 mol / l / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 0.2 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.05 mol / g / h to 0.2 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h;

[0121] 0.03 mol / g / h to 1.0 mol / g / h, 0.02 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.05 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.1 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.2 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.3 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.3 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.02 mol / g / h to 0.3 mol / g / h and 0 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.05 mol / g / h to 0.3 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.1 mol / g / h to 0.3 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.2 mol / g / h to 0.3 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0. 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.2 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.02 mol / g / h to 0.2 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.03 mol / g / h to 0.2 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.0.5 mol / g / h to 0.2 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.1 mol / g / h to 0.2 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.1 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1 0.0 mol / g / h, 0.02 mol / g / h to 0.1 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.03 mol / g / h to 0.1 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.05 mol / g / h to 0.1 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h;

[0122] 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.05 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.1 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.2 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.3 mol / g / h to 0.5 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.3 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0 0.02 mol / g / h to 0.3 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.05 mol / g / h to 0.3 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.1 mol / g / h to 0.3 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.2 mol / g / h to 0.3 mol / g / h; 0. 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.2 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.02 mol / g / h to 0.2 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.03 mol / g / h to 0.2 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.0.01 mol / g / h to 0.5 mol / g / h and 0.05 mol / g / h to 0.2 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.1 mol / g / h to 0.2 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.1 mol / g / h; 0.03 mol / g / h to 1 0.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h, and 0.02 mol / g / h to 0.1 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, 0.01 mol / g / h to 0.5 mol / g / h, and 0.03 mol / g / h to 0.1 mol / g / h; 0.03 mol / g / h to 1.0 mol / g / h, or 0.01 mol / g / h to 0.5 mol / g / h and 0.05 mol / g / h to 0.1 mol / g / h.

[0123] More preferably, 0.05 mol / g / h to 1.0 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h; 0.1 mol / g / h to 1.0 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h; 0.15 mol / g / h to 1. 0 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h; 0.05 mol / g / h to 0.5 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h; 0.1 ...2 mol / g / h to 0.5 mol / g / h; 0.02 mol / g / h to 0.5 mol / g / h; 0.02 mol / g / h to 0.5 mol / g / h; 0.02 mol / g / h to 0.5 mol / g / h; 0.02 mol / g / h to 0.5 mol / g / h; 0.02 mol / g / h to 0.5 mol / g / h; 0.02 mol / g / h to 0.5 mol / g / h; 0.02 mol / g / h to 0.5 mol / g / h 0.02 mol / g / h to 0.5 mol / g / h and 0.15 mol / g / h to 0.5 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h; 0.05 mol / g / h to 0.3 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h. 0.1 mol / g / h to 0.3 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h, and 0.02 mol / g / h to 0.5 mol / g / h; 0.15 mol / g / h to 0.3 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h, and 0.02 mol / g / h to 0.5 mol / g / h;

[0124] 0.05 mol / g / h to 1.0 mol / g / h, 0.2 mol / g / h to 0.5 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h; 0.1 mol / g / h to 1.0 mol / g / h, 0.2 mol / g / h to 0.5 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h; 0.15 mol / g / h to 1.0 mol / g / h g / h, 0.2 mol / g / h to 0.5 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h; 0.05 mol / g / h to 0.5 mol / g / h, 0.01 mol / g / h to 0.2 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h; 0.1 mol / g / h to 0.5 mol / g / h, 0.03 mol / g / h to 0 0.2 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h; 0.15 mol / g / h to 0.5 mol / g / h, 0.05 mol / g / h to 0.2 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h; 0.05 mol / g / h to 0.3 mol / g / h, 0.01 mol / g / h to 0.1 mol / g / h and 0.02 mol / g / h l / g / h to 0.5 mol / g / h; 0.1 mol / g / h to 0.3 mol / g / h, 0.03 mol / g / h to 0.1 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h; 0.15 mol / g / h to 0.3 mol / g / h, 0.05 mol / g / h to 0.1 mol / g / h and 0.02 mol / g / h to 0.5 mol / g / h;

[0125] 0.05 mol / g / h to 1.0 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h and 0.2 mol / g / h to 0.5 mol / g / h; 0.1 mol / g / h to 1.0 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h and 0.2 mol / g / h to 0.5 mol / g / h; 0.15 mol / g / h to 1.0 mol / h g / h, 0.03 mol / g / h to 0.5 mol / g / h and 0.2 mol / g / h to 0.5 mol / g / h; 0.05 mol / g / h to 0.5 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.2 mol / g / h; 0.1 mol / g / h to 0.5 mol / g / h, 0.03 mol / g / h to 0.01 mol / g / h to 0.2 mol / g / h; 0.1 mol / g / h to 0.5 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h and ...5 mol / g / h; 0.03 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h to 0.5 mol / g / h; 0.01 mol / g / h to 0.5 mol / g / h and 0.03 mol / g / h to 0.5 mol / g / h and 0.05 mol / g / h to 0.5 mol / g / h and 0.03 mol / g / h to 0.5 mol / g / h and 0.05 mol / g / h to 0.5 mol / g / h and 0.03 mol / g / h to 0.5 mol / g / h and 0.05 mol / g / h to 0.5 0.5 mol / g / h and 0.03 mol / g / h to 0.2 mol / g / h; 0.15 mol / g / h to 0.5 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h and 0.05 mol / g / h to 0.2 mol / g / h; 0.05 mol / g / h to 0.3 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h and 0.01 mol / g / h. l / g / h to 0.1 mol / g / h; 0.1 mol / g / h to 0.3 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h and 0.03 mol / g / h to 0.1 mol / g / h; 0.15 mol / g / h to 0.3 mol / g / h, 0.03 mol / g / h to 0.5 mol / g / h and 0.02 mol / g / h to 0.1 mol / g / h;

[0126] 0.1 mol / g / h to 1.0 mol / g / h, 0.05 mol / g / h to 0.5 mol / g / h and 0.05 mol / g / h to 0.5 mol / g / h; 0.1 mol / g / h to 1.0 mol / g / h, 0.1 mol / g / h to 0.5 mol / g / h and 0.1 mol / g / h to 0.5 mol / g / h; 0.4 mol / g / h to 1.0 mol / g / h, 0.1 mol / g / h to 0.3 mol / g / h and 0.1 mol / g / h to 0.3 mol / g / h; 0.05 mol / g / h to 0.5 mol / g / h, 0.0 3 mol / g / h to 0.15 mol / g / h, 0.02 mol / g / h to 0.1 mol / g / h; 0.1 mol / g / h to 1.0 mol / g / h, 0.08 mol / g / h to 0.36 mol / g / h and 0.06 mol / g / h to 0.26 mol / g / h; 0.08 mol / g / h to 0.75 mol / g / h, 0.06 mol / g / h to 0.27 mol / g / h and 0.05 mol / g / h to 0.2 mol / g / h; 0.08 mol / g / h to 0.5 mol / g / h, 0.06 mol / g / h to 0.18 mol / g / h / g / hour and 0.05mol / g / hour to 0.13mol / g / hour; 0.05mol / g / hour to 0.75mol / g / hour, 0.04mol / g / hour to 0.27mol / g / hour and 0.03mol / g / hour to 0.18mol / g / hour; 0.05mol / g / hour to 0.6mol / g / hour, 0.04mol / g / hour to 0.2mol / g / hour and 0.03mol / g / hour to 0.15mol / g / hour; 0.06mol / g / hour to 0.6mol / g / hour, 0.05mol / g / hour to 0.2mol / g / hour and 0.04mol / g / hour 0.06 mol / g / hour to 0.5 mol / g / hour, 0.05 mol / g / hour to 0.18 mol / g / hour and 0.04 mol / g / hour to 0.13 mol / g / hour; 0.05 mol / g / hour to 0.5 mol / g / hour, 0.04 mol / g / hour to 0.18 mol / g / hour and 0.03 mol / g / hour to 0.13 mol / g / hour; or 0.04 mol / g / hour to 0.4 mol / g / hour, 0.02 mol / g / hour to 0.1 mol / g / hour and 0.01 mol / g / hour to 0.1 mol / g / hour.

[0127] Gaseous substrate is typically bubbled into the bioreactor from the bottom. Microorganisms present in the bioreactor utilize the gaseous substrate to produce biomass. In this method, oxygen and hydrogen inputs are controlled as part of a feedback loop to allow gases initially added to the system above their explosion limits, as they are initially injected into the liquid culture medium phase. The feedback control then ensures that the system fully utilizes oxygen and / or hydrogen, such that gases causing headspace are kept below their explosion safety limits, which are 5% (v / v) for oxygen and 4% (v / v) for hydrogen under standard conditions. Furthermore, all three gas inputs are controlled such that oxygen or hydrogen remains the limiting gas within the system.

[0128] The control input flow includes maintaining the molar ratio of dissolved hydrogen:oxygen:carbon dioxide in the liquid phase at a distance of 0 mm to 750 mm from the gas phase in direct contact with the liquid phase, at 0 to 40:0 to 15:0 to 15, 0 to 12.748:0 to 4.25:0 to 2.0, 3.183 to 12.748:0 to 1.0625:0.75 to 2.0, 0 to 3.187:0.795 to 4.25:0.75 to 2.0, or 3.183 to 12.748:0.795 to 4.25:0 to 0.5.

[0129] Preferably, controlling the input flow includes maintaining the molar ratio of dissolved hydrogen:oxygen:carbon dioxide in the liquid phase at a distance of 0 mm to 750 mm from the gas phase in direct contact with the liquid phase, in the following ratios: 1 to 12.748:0 to 1.0625:0.75 to 2.0, 2 to 12.748:0 to 1.0625:0.75 to 2.0, 4 to 12.748:0 to 1.0625:0.75 to 2.0, 6 to 12.748:0 to 1.0625:0.75 to 2.0, 8 to 12.748:0 to 1.0625:0.75 to 2.0, 1 to 20:0 to 1.0625:0.75 to 2.0. 2.0, 2 to 20: 0 to 1.0625: 0.75 to 2.0, 4 to 20: 0 to 1.0625: 0.75 to 2.0, 6 to 20: 0 to 1.0625: 0.75 to 2.0, 8 to 20: 0 to 1.0625: 0.75 to 2.0, 12 to 20: 0 to 1.0625: 0.75 to 2.0, 1 to 30: 0 to 1.0625: 0.75 to 2.0, 2 to 30: 0 to 1.0625: 0.75 to 2.0, 4 to 30: 0 to 1.0625: 0.75 to 2.0, 6 to 30: 0 to 1.0625: 0.75 to 2.0, 8 to 3 0:0 to 1.0625:0.75 to 2.0, 12 to 30:0 to 1.0625:0.75 to 2.0, 20 to 30:0 to 1.0625:0.75 to 2.0, 1 to 40:0 to 1.0625:0.75 to 2.0, 2 to 40:0 to 1.0625:0.75 to 2.0, 4 to 40:0 to 1.0625:0.75 to 2.0, 6 to 40:0 to 1.0625:0.75 to 2.0, 8 to 40:0 to 1.0625:0.75 to 2.0, 12 to 40:0 to 1.0625:0.75 to 2.0, 20 to 40:0 To 1.0625:0.75 to 2.0, 1 to 8:0 to 1.0625:0.75 to 2.0, 2 to 8:0 to 1.0625:0.75 to 2.0, 4 to 8:0 to 1.0625:0.75 to 2.0, 6 to 8:0 to 1.0625:0.75 to 2.0, 1 to 6:0 to 1.0625:0.75 to 2.0, 2 to 6:0 to 1.0625:0.75 to 2.0, 4 to 6:0 to 1.0625:0.75 to 2.0, 1 to 4:0 to 1.0625:0.75 to 2.0, 2 to 4:0 to 1.0625:0.75 to 2.0,

[0130] 3.183 to 12.748:0 to 1.0625:0.25 to 2.0, 3.183 to 12.748:0 to 1.0625:0.5 to 2.0, 3.183 to 12.748:0 to 1.0625:1 to 2.0, 3.183 to 12.748:0 to 1.0625:0.25 to 2.5, 3.183 to 12.748:0 to 1.0625:0.5 to 2.5, 3.183 to 12.748:0 to 1.0625:1 to 2.5, 3.183 to 12.748:0 to 1.0625:2 to 2.5, 3.183 to 12.748:0 to 1.0 625:0.25 to 3.5, 3.183 to 12.748:0 to 1.0625:0.5 to 3.5, 3.183 to 12.748:0 to 1.0625:1 to 3.5, 3.183 to 12.748:0 to 1.0625:2 to 3.5, 3.183 to 12.748:0 to 1.0625:0.25 to 5, 3.183 to 12.748:0 to 1.0625:0.5 to 5, 3.183 to 12.748:0 to 1.0625:1 to 5, 3.183 to 12.748:0 to 1.0625:2 to 5, 3.183 to 12.748:0 to 1 .0625:3.5 to 5, 0.25 to 10, 3.183 to 12.748:0 to 1.0625:0.5 to 10, 3.183 to 12.748:0 to 1.0625:1 to 10, 3.183 to 12.748:0 to 1.0625:2 to 10, 3.183 to 12.748:0 to 1.0625:3.5 to 10, 3.183 to 12.748:0 to 1.0625:5 to 10, 3.183 to 12.748:0 to 1.0625:0.25 to 15, 3.183 to 12.748:0 to 1.0625:0.5 to 15, 3.183 to 12.748:0 to 1.0625:1 to 15, 3.183 to 12.748:0 to 1.0625:2 to 15, 3.183 to 12.748:0 to 1.0625:3.5 to 15, 3.183 to 12.748:0 to 1.0625:5 to 15, 3.183 to 12.748:0 to 1.0625:10 to 15, 3.183 to 12.748:0 to 1.0625:0.25 to 1.0, 3.183 to 12.748:0 to 1.0625:0.5 to 1.0, or 3.183 to 12.748:0 to 1.0625:0.75 to 1.0.

[0131] Preferably, controlling the input flow includes maintaining the molar ratio of hydrogen:oxygen:carbon dioxide dissolved in the liquid phase at a distance of 0 mm to 750 mm from the gas phase in direct contact with the liquid phase as follows: 0 to 3.187:0.25 to 4.25:0.75 to 2.0, 0 to 3.187:1.5 to 4.25:0.75 to 2.0, 0 to 3.187:2.5 to 4.25:0.75 to 2.0, 0 to 3.187:0.25 to 6:0.75 to 2.0, 0 to 3.187:0.795 to 6:0.75 to 2.0, 0 to 3. 187:1.5 to 6:0.75 to 2.0, 0 to 3. 187:2.5 to 6:0.75 to 2.0, 0 to 3. 187:0.25 to 10:0.75 to 2.0, 0 to 3. 187:0.795 to 10:0.75 to 2.0, 0 to 3. 187:1.5 to 10:0.75 to 2.0, 0 to 3. 187:2.5 to 10:0.75 to 2.0, 0 to 3. 187:5 to 10:0.75 to 2.0, 0 to 3. 187:0.25 to 15:0.75 to 2.0, 0 to 3. 187:0.795 to 15:0.75 to 2.0, 0 to 3. 187:1.5 to 15:0.75 to 2.0, 0 to 3. 187:2.5 to 15:0.75 to 2.0, 0 to 3. 187:5 to 15:0.75 to 2.0, 0 to 3. 187:10 to 15:0.75 to 2.0, 0 to 3. 187:0.25 to 3:0.75 to 2.0, 0 to 3. 187:0.795 to 3:0.75 to 2.0, 0 to 3. 187:1.5 to 3:0.75 to 2.0, 0 to 3.1 87:2.5 to 3:0.75 to 2.0, 0 to 3.187:0.25 to 2:0.75 to 2.0, 0 to 3.187:0.795 to 2:0.75 to 2.0, 0 to 3.187:1.5 to 2:0.75 to 2.0, 0 to 3.187:0.25 to 1.5:0.75 to 2.0, 0 to 3.187:0.795 to 1.5:0.75 to 2.0, 0 to 3.187:0.25 to 1:0.75 to 2.0, 0 to 3.187:0.795 to 1:0.75 to 2.0

[0132] 0 to 3.187:0.795 to 4.25:0.25 to 2.0, 0 to 3.187:0.795 to 4.25:0.5 to 2.0, 0 to 3.187:0.795 to 4.25:1 to 2.0, 0 to 3.187:0.795 to 4.25:0.25 to 2.5, 0 to 3.187:0.795 to 4.25:0.5 to 2.5, 0 to 3.187:0.795 to 4.25:1 to 2.5, 0 to 3.187:0.795 to 4.25:2 to 2.5, 0 to 3.187:0.795 to 4.25. 795 to 4.25: 0.25 to 3.5, 0 to 3.187: 0.795 to 4.25: 0.5 to 3.5, 0 to 3.187: 0.795 to 4.25: 1 to 3.5, 0 to 3.187: 0.795 to 4.25: 2 to 3.5, 0 to 3.187: 0.795 to 4.25: 0.25 to 5, 0 to 3.187: 0.795 to 4.25: 0.5 to 5, 0 to 3.187: 0 to 1.0625: 1 to 5, 0 to 3.187: 0.795 to 4.25: 2 to 5, 0 to 3.18 7:0.795 to 4.25:3.5 to 5, 0.25 to 10, 0 to 3.187:0.795 to 4.25:0.5 to 10, 0 to 3.187:0.795 to 4.25:1 to 10, 0 to 3.187:0.795 to 4.25:2 to 10, 0 to 3.187:0.795 to 4.25:3.5 to 10, 0 to 3.187:0.795 to 4.25:5 to 10, 0.25 to 15, 0 to 3.187:0.795 to 4.25:0.5 to 15, 0 to 3.187:0.7 95 to 4.25:1 to 15, 0 to 3.187:0.795 to 4.25:2 to 15, 0 to 3.187:0.795 to 4.25:3.5 to 15, 0 to 3.187:0.795 to 4.25:5 to 15, 0 to 3.187:0.795 to 4.25:10 to 15, 0 to 3.187:0.795 to 4.25:0.25 to 1.0, 0 to 3.187:0.795 to 4.25:0.5 to 1.0, or 0 to 3.187:0.795 to 4.25:0.75 to 1.0.

[0133] Preferably, controlling the input flow includes maintaining a molar ratio of hydrogen:oxygen:carbon dioxide dissolved in the liquid phase of 3.183 to 12.748:0.795 to 4.25:0 to 0.5 within a distance of 0 mm to 750 mm from the gas phase in direct contact with the liquid phase.

[0134] 1 to 12.748: 0.795 to 4.25: 0 to 0.5, 2 to 12.748: 0.795 to 4.25: 0 to 0.5, 4 to 12.748: 0.795 to 4.25: 0 to 0.5, 6 to 12.748: 0.795 to 4.25: 0 to 0.5, 8 to 12.748: 0.795 to 4.25: 0 to 0.5, 1 to 20: 0.795 to 4.25: 0 to 0.5, 2 to 20: 0.795 to 4.25: 0 to 0.5, 4 to 20: 0.795 to 4.25: 0 0.5, 6 to 20: 0.795 to 4.25: 0 to 0.5, 8 to 20: 0.795 to 4.25: 0 to 0.5, 12 to 20: 0.795 to 4.25: 0 to 0.5, 1 to 30: 0.795 to 4.25: 0 to 0.5, 2 to 30: 0.795 to 4.25: 0 to 0.5, 4 to 30: 0.795 to 4.25: 0 to 0.5, 6 to 30: 0.795 to 4.25: 0 to 0.5, 8 to 30: 0.795 to 4.25: 0 to 0.5, 12 to 30: 0.7 95 to 4.25: 0 to 0.5, 20 to 30: 0.795 to 4.25: 0 to 0.5, 1 to 40: 0.795 to 4.25: 0 to 0.5, 2 to 40: 0.795 to 4.25: 0 to 0.5, 4 to 40: 0.795 to 4.25: 0 to 0.5, 6 to 40: 0.795 to 4.25: 0 to 0.5, 8 to 40: 0.795 to 4.25: 0 to 0.5, 12 to 40: 0.795 to 4.25: 0 to 0.5, 20 to 40: 0.795 to 4.25: 0 to 0. 5. 1 to 8: 0.795 to 4.25: 0 to 0.5; 2 to 8: 0.795 to 4.25: 0 to 0.5; 4 to 8: 0.795 to 4.25: 0 to 0.5; 6 to 8: 0.795 to 4.25: 0 to 0.5; 1 to 6: 0.795 to 4.25: 0 to 0.5; 2 to 6: 0.795 to 4.25: 0 to 0.5; 4 to 6: 0.795 to 4.25: 0 to 0.5; 1 to 4: 0.795 to 4.25: 0 to 0.5; 2 to 4: 0.795 to 4.25: 0 to 0.5.

[0135] 3.183 to 12.748: 0.25 to 4.25: 0 to 0.5, 3.183 to 12.748: 1.5 to 4.25: 0 to 0.5, 3.183 to 12.748: 2.5 to 4.25: 0 to 0.5, 3.183 to 12.748: 0.25 to 6: 0 to 0.5, 3.183 to 12.748: 0.795 to 6: 0 to 0.5, 3.183 to 12.748: 1.5 to 6: 0 to 0.5, 3.1 83 to 12.748: 0.25 to 10: 0 to 0.5, 3.183 to 12.748: 0.795 to 10: 0 to 0.5, 3.183 to 12.748: 1.5 to 10: 0 to 0.5, 3.183 to 12.748: 2.5 to 10: 0 to 0.5, 3.183 to 12.748: 5 to 10: 0 to 0.5, 3.183 to 12.748: 0.25 to 15: 0 to 0.5, 3.183 to 12.748: 0.795 to 15: 0 to 0.5, 3.183 to 12. 748:1.5 to 15:0 to 0.5, 3.183 to 12.748:2.5 to 15:0 to 0.5, 3.183 to 12.748:5 to 15:0 to 0.5, 3.183 to 12.748:10 to 15:0 to 0.5, 3.183 to 12.748:0.25 to 3:0 to 0.5, 3.183 to 12.748:0.795 to 3:0 to 0.5, 3.183 to 12.748:1.5 to 3:0 to 0.5, 3.183 to 12.748:2.5 to 3:0 to 0 0.5, 3.183 to 12.748: 0.25 to 2: 0 to 0.5, 3.183 to 12.748: 0.795 to 2: 0 to 0.5, 3.183 to 12.748: 1.5 to 2: 0 to 0.5, 3.183 to 12.748: 0.25 to 1.5: 0 to 0.5, 3.183 to 12.748: 0.795 to 1.5: 0 to 0.5, 3.183 to 12.748: 0.25 to 1: 0 to 0.5, or 3.183 to 12.748: 0.795 to 1: 0 to 0.5.

[0136] As described above, controlling the input flow includes maintaining the molar ratio of hydrogen:oxygen:carbon dioxide dissolved in the liquid phase within a distance of 0 mm to 750 mm from the gas phase in direct contact with the liquid phase. More preferably, this distance includes 0 mm to 700 mm, 0 mm to 650 mm, 0 mm to 600 mm, 0 mm to 550 mm, 0 mm to 500 mm, 0 mm to 450 mm, 0 mm to 400 mm, 0 mm to 350 mm, 0 mm to 300 mm, 0 mm to 250 mm, 0 mm to 200 mm, 0 mm to 150 mm, 0 mm to 100 mm, 0 mm to 90 mm, 0 mm to 80 mm, 0 mm to 70 mm, 0 mm to 60 mm, 0 mm to 50 mm, 0 mm to 40 mm, 0 mm to 30 mm, 0 mm to 20 mm, or 0 mm to 10 mm.

[0137] The method according to the invention is preferably performed using a bioreactor suitable for an industrial environment. Preferably, the bioreactor according to the invention is a chemostat.

[0138] Bioreactors suitable for industrial environments adhere to standard requirements in the industrial fermentation field using microorganisms (preferably hydrogen-oxidizing microorganisms). Bioreactors suitable for industrial environments typically have a capacity of approximately 0.2 m³ for pilot-scale production. 3 Approximately 10m 3 The liquid phase volume and approximately 2m³ for factory-scale bioreactors 3 Approximately 500m 3 The liquid phase volume is [amount], but theoretically there is no upper limit to the volume. Standard requirements in the field of industrial fermentation of microorganisms preferably include the ability to withstand high thermal stress and / or high internal gas and liquid pressures. Standard requirements according to the invention include design considerations to prevent and / or withstand the consequences of potentially explosive gas mixtures containing hydrogen and oxygen. Therefore, the bioreactors according to the invention suitable for industrial environments enable the use of high-concentration gaseous substrates, such as hydrogen and / or oxygen, according to the invention.

[0139] In the context of this invention, a bioreactor, preferably a chemostat, can be used to maintain the physiological state and specific growth rate of microorganisms substantially constant. This is achieved by maintaining various continuous (bio)chemical processes, such as controlling stirring speed, gaseous substrate transfer rate, dilution rate (volume flow rate of supplied nutrients divided by total volume), temperature, pH, removing culture medium containing microorganisms, and / or adding culture medium to preferably maintain a substantially constant liquid culture volume. For example, the specific growth rate of microorganisms can be controlled by varying the rate at which culture medium is added to the bioreactor. Increasing the dilution rate will increase microbial growth. However, it is necessary to control the dilution rate relative to the specific growth rate to prevent wash-out. Controlling the dilution rate is to maximize the protein production rate and protein content of the microorganisms. When the specific growth rate of microorganisms is too high, the protein content and / or quality of the biomass may decrease.

[0140] The bioreactor used to perform the method according to the invention is preferably selected from the group consisting of bubble column reactors, air-lift reactors, continuous stirred tank reactors, and circulating reactors. A preferred bioreactor for performing the method according to the invention is a continuous stirred tank reactor. Preferably, the bioreactor includes one or more gas and / or liquid circulation systems.

[0141] The principle behind achieving higher levels of protein content and quality in biomass through liquid phase replacement within a bioreactor is maintaining microorganisms in a physiologically stable state under constant environmental conditions. In this stable state, growth occurs at a substantially constant specific growth rate, and all culture parameters, such as pH, nutrient concentration, gaseous substrate concentration, and microbial concentration, remain substantially constant. Such physiological stability of the microorganisms has been found essential for producing biomass containing at least 65% protein at rates greater than 10 g / L / day. Therefore, according to the invention, it is preferable to control the input flow and nutrient composition to maintain the specific growth rate and / or stable state of the microorganisms, more preferably the stable state of the microorganisms, and even more preferably the specific growth rate of the microorganisms.

[0142] According to the present invention, it is preferred to control the input flow and nutrient composition to achieve or maintain a stable state of microorganisms by maintaining a microbial concentration in the liquid phase of the bioreactor at at least 5 g / L, preferably from 5 g / L to 100 g / L. More preferably, the microbial concentration in the liquid phase of the bioreactor can be maintained at at least 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L to 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 25 g / L, or 30 g / L, preferably 6 g / L or 7 g / L. 8 g / l, 9 g / l, 10 g / l to 11 g / l, 12 g / l, 13 g / l, 14 g / l, 15 g / l, 16 g / l, 17 g / l, 18 g / l, 19 g / l, 20 g / l, 25 g / l or 30 g / l to 50 g / l, 60 g / l, 70 g / l, 80 g / l, 90 g / l or 100 g / l to achieve or maintain the stable state of microorganisms. Even more preferably, the stable state of the microorganisms can be achieved or maintained by maintaining the concentration of microorganisms in the liquid phase of the bioreactor at at least 8 g / L, 9 g / L, 10 g / L to 11 g / L, 12 g / L, 13 g / L, 14 g / L or 15 g / L, preferably 8 g / L, 9 g / L, 10 g / L to 11 g / L, 12 g / L, 13 g / L, 14 g / L or 15 g / L to 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L or 50 g / L.

[0143] Preferably, controlling the input flow and nutrient composition according to the present invention includes maintaining a specific growth rate of microorganisms of at least 1.0 / day, preferably at least 1.1 / day, 1.2 / day, 1.3 / day, 1.4 / day, 1.5 / day, 1.6 / day, 1.7 / day, 1.8 / day, 1.9 / day, 2.0 / day, 2.1 / day, 2.2 / day, 2.3 / day, 2.4 / day, 2.5 / day, 2.6 / day, 2.7 / day, 2.8 / day, 2.9 / day, 3.0 / day, 3.1 / day, 3.2 / day, 3.3 / day, 3.4 / day, 3.5 / day, 3.6 / day, 3.7 / day, 3.8 / day, 3.9 / day, or 4.0 / day. More preferably, according to the invention, controlling the input flow and nutrient composition includes maintaining a specific growth rate of microorganisms of at least 1.5 / day, 1.6 / day, 1.7 / day, 1.8 / day, 1.9 / day, 2.0 / day, 2.1 / day, 2.2 / day, 2.3 / day, 2.4 / day, or 2.5 / day. Preferably, controlling the input flow and nutrient composition according to the present invention includes maintaining a specific growth rate of microorganisms of at least 0.03 / hour, preferably at least 0.03 / hour, 0.04 / hour, 0.05 / hour, 0.06 / hour, 0.07 / hour, 0.08 / hour, 0.09 / hour, 0.1 / hour, 0.11 / hour, 0.12 / hour, 0.13 / hour, 0.14 / hour, 0.15 / hour, 0.16 / hour, 0.17 / hour, 0.18 / hour, 0.2 / hour, 0.21 / hour, 0.22 / hour, 0.23 / hour, 0.24 / hour, 0.25 / hour, or 0.3 / hour. More preferably, according to the invention, controlling the input flow and nutrient composition includes maintaining a specific growth rate of microorganisms of at least 0.05 / hour, 0.06 / hour, 0.07 / hour, 0.08 / hour, 0.09 / hour, or 0.1 / hour.

[0144] When the specific growth rate of microorganisms is too high, the protein content and / or quality of biomass may decrease. For example, the nucleic acid content of biomass may become too high.

[0145] Therefore, preferably, controlling the input flow and nutrient composition according to the present invention includes maintaining a specific growth rate of microorganisms at 1.0 / day to 8.0 / day. More preferably, controlling the input flow and nutrient composition includes maintaining a specific growth rate of microorganisms at 1.1 / day, 1.2 / day, 1.3 / day, 1.4 / day, 1.5 / day, 1.6 / day, 1.7 / day, 1.8 / day, 1.9 / day, or 2.0 / day to 3.0 / day, 3.1 / day, 3.2 / day, 3.3 / day, 3.4 / day, 3.5 / day, 3.6 / day, 3.7 / day, 3.8 / day, 3.9 / day, 4.0 / day, 4.5 / day, 5.0 / day, 5.5 / day, 6.0 / day, or 7.0 / day. Even more preferably, controlling the input flow and nutrient composition includes maintaining a specific growth rate of microorganisms at 1.5 / day, 1.6 / day, 1.7 / day, 1.8 / day, 1.9 / day, 2.0 / day, 2.1 / day, 2.2 / day, 2.3 / day, 2.4 / day to 2.6 / day, 2.7 / day, 2.8 / day, 2.9 / day, 3.0 / day, 3.1 / day, 3.2 / day, 3.3 / day, 3.4 / day, or 3.5 / day. Preferably, controlling the input flow and nutrient composition according to the invention includes maintaining a specific growth rate of microorganisms at 0.03 / hour to 0.4 / hour. More preferably, controlling the input flow and nutrient composition includes maintaining the specific growth rate of the microorganisms at 0.04 / hour, 0.05 / hour, 0.06 / hour, 0.07 / hour, 0.08 / hour, 0.09 / hour or 0.1 / hour to 0.11 / hour, 0.12 / hour, 0.13 / hour, 0.14 / hour, 0.15 / hour, 0.16 / hour, 0.17 / hour, 0.18 / hour, 0.19 / hour, 0.2 / hour, 0.25 / hour, 0.3 / hour or 0.35 / hour. Even more preferably, controlling the input flow and nutrient composition includes maintaining the specific growth rate of the microorganisms at 0.05 / hour, 0.06 / hour, 0.07 / hour, 0.08 / hour, or 0.09 / hour to 0.11 / hour, 0.12 / hour, 0.13 / hour, 0.14 / hour, 0.15 / hour, 0.16 / hour, 0.17 / hour, 0.18 / hour, 0.19 / hour, or 0.2 / hour.

[0146] The applicant has unexpectedly discovered that bacteria selected from the genus *Alcaligenes* can produce high-quality biomass at high production rates. Therefore, this invention relates to a method for producing biomass from bacteria selected from the genus *Alcaligenes*. The applicant has also unexpectedly discovered that bacteria selected from the genus *Copper-loving Bacteria* can produce even higher-quality biomass at even higher production rates. Therefore, this invention relates to a method for producing biomass from bacteria selected from the preferred species of *Copper-loving Bacteria*, *Hookworm Copper-loving Bacteria*. Preferably, the biomass comprises at least 65% protein by dry weight of the total biomass. Preferably, the biomass is produced at a rate greater than 10 g / L / day. Preferably, the method of producing biomass includes using one or more input streams containing one or more gaseous substrates, said gaseous substrates comprising hydrogen, oxygen, and / or carbon dioxide, said method including contacting microorganisms in a liquid phase with a nutrient composition comprising compounds containing carbon, nitrogen, and / or phosphorus, and the gaseous substrates, wherein the input streams and nutrient compositions are controlled. This invention also relates to a method for producing biomass from bacteria selected from the genus *Alcaligenes*, isolating the produced biomass, and removing the nutrient composition, said method including downstream processing. The present invention also relates to a method for producing biomass from bacteria selected from the preferred species of the genus *Hookworm*, isolating the produced biomass and removing nutrients from the composition, the method comprising downstream processing.

[0147] To further utilize the produced biomass as a nutrient source for other organisms, it is necessary to process the biomass. In the agri-food industry, the removal of nutrient compositions and water components from the produced biomass is typically carried out. To facilitate transportation, storage, and prevent contamination by pathogens or other undesirable organisms, the water content of the produced biomass for further application needs to be as low as possible. Therefore, the present invention also relates to a method for separating biomass produced according to the method of the present invention and removing nutrient compositions, the method comprising downstream processing. Furthermore, the present invention relates to a method for separating biomass produced according to the present invention and removing nutrient compositions, the method comprising dehydrating and / or drying the biomass such that the biomass contains less than 10% by weight of water. Preferably, the biomass contains less than 9.0 wt%, 8.0 wt%, 7.0 wt%, 6.5 wt%, 6.0 wt%, 5.5 wt%, 5.0 wt%, 4.5 wt%, 4.0 wt%, 3.5 wt%, 3.0 wt%, 2.5 wt%, 2.0 wt%, 1.5 wt%, 1.0 wt%, or 0.5 wt% water.

[0148] Nutrient compositions obtained by separating biomass produced according to the method of the present invention and removing the nutrient composition (including downstream processing) or by separating biomass produced according to the present invention and removing the nutrient composition (including dehydrating and / or drying the biomass so that the biomass contains less than 10 wt%, 9.0 wt%, 8.0 wt%, 7.0 wt%, 6.5 wt%, 6.0 wt%, 5.5 wt%, 5.0 wt%, 4.5 wt%, 4.0 wt%, 3.5 wt%, 3.0 wt%, 2.5 wt%, 2.0 wt%, 1.5 wt%, 1.0 wt%, or 0.5 wt%) can be used as nutrient compositions for producing biomass according to the method of the present invention.

[0149] Biomass produced or obtained by any method of this disclosure can be used to feed or provide nutrition to one or more organisms. Similar feeds are commonly used in the agricultural industry for organisms such as fish, crustaceans, mollusks, poultry, pigs, and cattle. Therefore, the present invention also relates to the use of biomass produced or obtained by any method of this disclosure for feeding or providing nutrition to, for example, fish, crustaceans, mollusks, poultry, pigs, and cattle. Preferably, fish include Cyprinidae, Salmonidae, Thunnini, Oreochromis, and Siluriformes. Preferably, poultry includes chickens (Gallus gallus domesticus).

[0150] The properties of biomass produced by chemoautotrophic bacteria are directly related to its value for certain applications. For example, for animal feed and food applications, protein content and amino acid composition are crucial to nutritional quality. The method of this invention produces such biomass with high nutritional quality. Therefore, the present invention also relates to biomass containing proteins obtainable by the method of the present invention, said proteins comprising the following amino acid contents: 0.6% to 6.4% histidine of total biomass dry weight protein content, 1.3% to 9.2% isoleucine of total biomass dry weight protein content, 2.5% to 16.0% leucine of total biomass dry weight protein content, 2.0% to 14.8% lysine of total biomass dry weight protein content, 0.7% to 7.2% methionine of total biomass dry weight protein content, 1.2% to 11.4% phenylalanine of total biomass dry weight protein content, 1.1% to 9.2% threonine of total biomass dry weight protein content, 0.3% to 5.2% tryptophan of total biomass dry weight protein content, and 1.1% to 12.4% valine of total biomass dry weight protein content. Preferably, the amino acid content includes 0.9% to 4.8% histidine, 2.0% to 6.9% isoleucine, 3.8% to 12.0% leucine, 3.0% to 11.1% lysine, 1.1% to 5.4% methionine, 1.7% to 8.5% phenylalanine, 1.6% to 6.9% threonine, 0.4% to 3.9% tryptophan, and 1.7% to 9.3% valine, based on the total biomass dry weight protein content. More preferably, the amino acid content includes 1.2% to 3.2% histidine, 2.6% to 4.6% isoleucine, 5.0% to 8.0% leucine, 4.0% to 7.4% lysine, 1.4% to 3.6% methionine, 2.3% to 5.7% phenylalanine, 2.1% to 4.6% threonine, 0.5% to 2.6% tryptophan, and 2.2% to 6.2% valine, based on the total biomass dry weight protein content.

[0151] Furthermore, the present invention relates to biomass obtainable by the method of the present invention, said biomass comprising a lipid content of 1.5% to 24% of the total dry weight of biomass, said lipid content comprising the following fatty acid contents: 15% to 80% of the total dry weight of biomass fatty acids of C16:O palmitic acid, 2.5% to 30% of the total dry weight of biomass fatty acids of C16:1 palmitoleic acid, and 15% to 80% of the total dry weight of biomass fatty acids of C17:1 heptadecanoic acid. Preferably, the lipid content comprises 2.3% to 18% of the total dry weight of biomass, said lipid content comprising the following fatty acid contents: 23% to 60% of the total dry weight of biomass fatty acids of C16:O palmitic acid, 3.8% to 22.3% of the total dry weight of biomass fatty acids of C16:1 palmitoleic acid, and 23% to 60% of the total dry weight of biomass fatty acids of C17:1 heptadecanoic acid. More preferably, the lipid content accounts for 3% to 12% of the total biomass dry weight, said lipid content includes the following fatty acid contents: 30% to 40% of the total biomass dry weight fatty acid content of C16:0 palmitic acid, 5% to 15% of the total biomass dry weight fatty acid content of C16:1 palmitoleic acid, and 30% to 40% of the total biomass dry weight fatty acid content of C17:1 heptadecanoic acid. Attached Figure Description

[0153] The invention will now be discussed with reference to the accompanying drawings, which illustrate preferred exemplary embodiments of the invention.

[0154] Figure 1 A preferred embodiment of the invention is illustrated. In this document, in the reactor flow diagram, the numbers represent the following features:

[0155] The oxygen / air input (1), hydrogen input (2), and carbon dioxide input (3) are controlled by a feedback loop based on analysis of their concentrations in the bioreactor via through-gas analysis and / or by a defined and adjustable input gas ratio for optimal protein production metabolism. There are also pH and OD-based feedback loops for the addition of inorganic nitrogen (e.g., urea) per unit of biomass produced (4) and for maintaining pH by adding a pH buffer (5). Liquid growth medium (6) is added to the bioreactor as needed in response to microbial growth measured by sensors in the various feedback loops. Unused gases can be recycled (7) back into the bioreactor. Another feature, the dilution rate, allows for a certain duration of maintenance in the reactor, optimal microbial growth, and optimal biomass production. The dilution rate is determined by controlling the input of inorganic nitrogen (4), pH buffer (5), liquid growth medium (6), and the recycling (10) of the removed biomass-containing liquid and the output of the removed biomass-containing liquid (8). The removal of the biomass-containing liquid (8) is followed by a downstream treatment step involving dehydration, in which a majority of the liquid is separated from the biomass-containing liquid. This majority of the liquid is then preferably recycled (10) to the bioreactor, while the biomass, along with a small portion of the remaining liquid, undergoes further downstream treatment steps (9) including further dehydration, drying, and microbial inactivation, which, through optional additional downstream treatment steps (11), ultimately yields a biomass product preferably suitable as a nutrient source for other organisms. Increased overall system productivity can be achieved by enabling higher oxygen concentrations throughout the system, thereby reducing oxygen limitations. A responsive oxygen feed (1) is preferred to increase reactor loading while maintaining a safe gas mixture concentration in the headspace. Oxygen in the system headspace is preferably maintained at less than 5%, but added in increased amounts in response to increased microbial gas consumption to achieve an optimal balance between protein production and growth rates. Maximum protein yield requires optimal availability of inorganic nitrogen compounds such as ammonia and / or urea in the system. Optimal growth rates can also be achieved by feeding ammonia (4) in response to defined process conditions.

[0156] Fine-grained control of all parameters together allows for increased overall fermentation productivity by reducing and optimizing substrate limitations. Even higher productivity can be achieved without these limitations, and crucially, control aspects have been developed to balance this increased productivity with the optimization of the protein content of the biomass.

[0157] Figure 2 Examples are shown of the range of total protein content and essential amino acid amounts per 100g of isolated biomass produced according to the method of the present invention.

[0158] Figure 3 An example is shown of the proportion of the most abundant fatty acid in the total fatty acid content of isolated biomass produced according to the method of the present invention. The fatty acid content of isolated biomass produced according to the method of the present invention is determined by the following method:

[0159] The sample was heated under reflux for 2 hours with a mixture of methanol and sulfuric acid in toluene. The fats and oils were transesterified to fatty acid methyl esters (FAMES). The resulting methyl ester mixture was extracted with a small amount of n-hexane. The n-hexane solution was then dried with anhydrous sodium sulfate, and the sample was transferred to a chromatographic vial. FAMES fatty acid profiles were obtained by gas-liquid chromatography using an FFAP column (25 m × 0.20 mm ID) and detected by a flame ionization detector. Profiles can be reported with or without an internal standard (C17:0).

[0160] Figure 4 The data shows the range of essential amino acid amounts compared to typical soybean meal and fishmeal used in animal agriculture (see, for example, the U.S. Soybean Meal Information Leaflet published by the U.S. Soybean Export Council, April 20, 2020, available online). https: / / ussec.org / wp-content / uploads / 2015 / 10 / US- Soybean-Meal-Information.pdf ; and fishmeal, see, for example, M. Das and SK Mandal; Oxya hylahyla (Orthoptera: Acrididae) as an Alternative Protein Source for Japanese Quail; International Scholarly Research Notices, 2014) , examples of the range of essential amino acid amounts per 100g of isolated biomass produced according to the method of the present invention. The amino acid content of isolated biomass produced according to the method of the present invention is determined by the following method:

[0161] The sample was oxidized in a combination of phenol, hydrogen peroxide, and formic acid. The oxidized sample was then hydrolyzed with hydrochloric acid. Amino acids were subsequently separated using ion-exchange chromatography and determined spectrophotometrically by their post-column reaction with ninhydrin.

[0162] It is clear that the biomass produced by the method according to the present invention has excellent quality.

[0163] Figure 5A and Figure 5BThe predicted biomass production rates are shown for different percentages of input oxygen and hydrogen concentrations. Oxygen and hydrogen concentrations are those added to the liquid phase, and the biomass production rate is the production rate of biomass with at least 65% protein, produced by the corresponding percentage of oxygen (…). Figure 5A ) or hydrogen ( Figure 5B A specific concentration percentage or higher causes a biomass production rate greater than 10 g / L / day. Figure 5A The amount of substrate other than oxygen is limited so that it is provided in the minimum amount sufficient for microbial viability. Figure 5B The amount of substrate other than hydrogen is limited so that it is provided in the minimum amount sufficient for microbial viability.

[0164] Figure 6 The invention illustrates a prediction of the protein content, by dry weight, of total biomass produced from hydrogen-oxidizing microorganisms, wherein controlling the input stream comprises adding hydrogen:oxygen in a molar ratio of 1:1 to 10:1 in the liquid phase, such that the biomass production rate is greater than 10 g / L / day.

[0165] Figure 7 The invention illustrates a prediction of the protein content, on a dry weight basis, of total biomass produced from hydrogen-oxidizing microorganisms, wherein controlling the input flow includes controlling the specific growth rate such that the biomass production rate is greater than 10 g / L / day.

[0166] Figure 8A A prediction of a preferred hydrogen transfer rate associated with the biomass production rate produced according to the invention is shown, wherein the biomass comprises at least 65% protein of the total biomass on a dry weight basis. The hydrogen transfer rate required for the target productivity can be calculated by multiplying the output of hydrogen relative to the biomass (Y_{H2 / X}) by the productivity, where Y_{H2 / X} is the number of grams of hydrogen used per gram of biomass formed. Figure 8A The necessary mass transfer was determined to achieve a productivity of more than 10 g / L / day (which is achievable using the method according to the invention) within an economically preferred range of hydrogen metabolic yields.

[0167] Figure 8B A prediction of a preferred oxygen transfer rate associated with the biomass production rate produced according to the invention is shown, wherein the biomass comprises at least 65% protein of the total biomass on a dry weight basis. The hydrogen transfer rate required for the target productivity can be calculated by multiplying the oxygen output relative to the biomass (Y_{O2 / X}) by the productivity, where Y_{O2 / X} is the number of grams of oxygen used per gram of biomass formed. Figure 8BThe necessary mass transfer required to achieve a productivity of more than 10 g / L / day (which is achievable using the method according to the invention) within an economically preferred range of oxygen metabolic yields was determined.

[0168] The following non-limiting embodiments illustrate the processes and materials according to the present invention.

[0169] Example 1

[0170] In such Figure 1 The industrial chemostat bioreactor system described herein cultivates Cupriavidis necator strain H16, also known as DSM 428 (Little et al.: "Complete Genome Sequence of Cupriavidis necator H16 (DSM 428)"; Microbiol. Resour. Announc. (2019)) or previously known as Ralstonia eutrophia H16 (Pohlmann et al.: "Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstoniaeutrophia H16"; Nature Biotechnology (2006)).

[0171] The DSMZ mineral medium 81(H-3) for chemo-inorganic nutrient growth was used, which was composed of and prepared as follows:

[0172] Solution A:

[0173] 2.3 g KH₂PO₄, 2.9 g Na₂HPO₄×2H₂O, 50 ml distilled water

[0174] Solution B:

[0175] NH4Cl 1.0g, MgSO4×7H2O 0.50g, CaCl2×2H2O 0.01g, MnCl2×4H2O 0.005g, NaVO3×H2O 0.005g, trace element solution SL-65ml, distilled water 915ml

[0176] Solution C:

[0177] Ferric ammonium citrate 0.05g, distilled water 20ml

[0178] Solutions A, B, and C were each heat-pressed at 121°C for 15 minutes, cooled to 50°C, and then aseptically mixed with 5.0 ml of a standard vitamin solution that had been sterilized by a filter (see below). The pH of the culture medium was adjusted to a range of 1 to 4, and then supplemented with an additional 1.5 g / L ammonium chloride and 3 × 10⁻⁶ ppm. -4 g / L NiCl2×6H2O and 1.5×10 -3 g / L ZnSO4×7H2O, and 1.5×10 -4 Supplemented with 0.15 g / L CuCl2×2H2O and 0.15 g / L ferric ammonium citrate. Using the above culture medium and preparation method, it is possible to support operating concentrations and productivity greater than 10 g / L / day of biomass (containing more than 65% protein) under gas-limited growth conditions, with in-situ pH control using a suitable alkali such as 0.2 M NaOH or NH4OH. To support even higher operating cell concentrations and productivity, the culture medium composition can be modified proportionally.

[0179] Standard vitamin solution:

[0180] Riboflavin 10mg, Thiamine-HCl×2H2O 50mg, Niacin 50mg, Pyridoxine-HCl 50mg, Ca-Pantothenate 50mg, Biotin 0.1mg, Folic Acid 0.2mg, Vitamin B12 1.0mg, Distilled Water 100ml

[0181] Trace element solution SL-6:

[0182] ZnSO4×7H2O 0.10g, MnCl2×4H2O 0.03g, H3BO3 0.30g, CoCl2×6H2O 0.20g, CuCl2×2H2O 0.01g, NiCl2×6H2O 0.02g, Na2MoO4×2H2O 0.03g, distilled water 1000ml

[0183] Allow the culture medium to grow for at least 3 days with constant stirring. Add carbon dioxide gas to the liquid phase at a non-limiting concentration. Add hydrogen and oxygen to the liquid phase at a ratio of 1:1 to 10:1. Optionally, recycle the gaseous substrate. Recycle the liquid culture medium. Add nutrient compounds and culture medium and remove a portion of the liquid phase from the system during culture. Keep the total volume of the liquid phase approximately constant. The gas transfer rate is determined according to... Figure 8A and Figure 8B The preferred rate is indicated.

[0184] According to the present invention, biomass is produced and separated to obtain a product having Figure 2 amino acid content and Figure 3The fatty acid content of biomass is shown in Table 1 below.

[0185] Table 1:

[0186]

[0187] Example 2

[0188] Using the culture conditions described in Example 1, as in Figure 1 Hookworm copper-loving bacteria strains were cultured in the industrial bioreactor system. Figure 5A The dilution rate is 2 / day. Figure 5B The dilution rate is 1 / day.

[0189] The result is as follows Figure 5A and Figure 5B The productivity, as shown and inferred from experimental results, is correlated with different hydrogen / oxygen inputs under constraints. Protein content is at least 65%.

[0190] Example 3

[0191] Using the culture conditions described in Example 1, as in Figure 1 The hookworm copper-loving bacteria strain was cultured in the industrial bioreactor system. The dilution rate was 1.87 / day.

[0192] The result is as follows Figure 6 The protein content, as shown and inferred from the experimental results, is correlated with different hydrogen:oxygen input ratios. The biomass productivity is at a rate greater than 10 g / L / day.

[0193] Example 4

[0194] Using the culture conditions described in Example 1, as in Figure 1 The hookworm copper-loving bacteria strain was cultured in the industrial bioreactor system. The dilution rates at the three points, from left to right, were 1.82 / day, 2.70 / day, and 3.53 / day, respectively. The H2:O2 ratio was 2:1.

[0195] The result is as follows Figure 7 The protein content and different specific growth rates are shown and inferred from the experimental results. The biomass productivity is a rate greater than 10 g / L / day.

[0196] Example 5

[0197] Using the culture conditions described in Example 1, as in Figure 1 Hookworm copper-loving bacteria strains were cultured in the industrial bioreactor system.

[0198] The result is as follows Figure 8A The productivity and different hydrogen transfer rates are shown in the table below, and inferred from the experimental results. Protein content is at least 65%.

[0199] Example 6

[0200] Using the culture conditions described in Example 1, as in Figure 1 Hookworm copper-loving bacteria strains were cultured in the industrial bioreactor system.

[0201] The result is as follows Figure 8B The productivity and different oxygen transfer rates are shown and inferred from the experimental results. The protein content is at least 65%.

Claims

1. A method for producing biomass from hydrogen-oxidizing microorganisms using one or more input streams containing one or more gaseous substrates, said biomass comprising at least 65% protein by dry weight of total biomass, said gaseous substrates comprising hydrogen and oxygen, said method comprising contacting said microorganisms in a liquid phase with a nutrient composition comprising nitrogen- and phosphorus-containing compounds and said gaseous substrates, wherein said one or more gaseous substrates comprise carbon dioxide or said nutrient composition comprises carbon-containing compounds, wherein said input streams and nutrient composition are controlled and wherein said biomass is produced at a rate greater than 10 g / L / day. Controlling the input flow includes adding hydrogen to oxygen in a molar ratio of 1:1 to 10:1 to the liquid phase. The hydrogen-oxidizing microorganisms mentioned above include bacteria from the species *Cupriavidus necator*; and The biomass is produced in a bioreactor suitable for an industrial environment, preferably wherein the liquid volume of the bioreactor suitable for an industrial environment is 0.2 to 10 m³. 3 For pilot-scale bioreactors, the size ranges from 2 to 500 m³. 3 Or more.

2. The method of claim 1, wherein the input flow and nutrient composition are controlled to maintain a specific growth rate and / or a stable state of the microorganisms.

3. The method according to claim 1 or 2 further comprises replacing the liquid phase of the bioreactor in which microorganisms grow and are maintained at 4 to 30% of the volume per hour.

4. The method according to any one of claims 1 to 3, wherein controlling the input flow and nutrient composition comprises maintaining a specific growth rate of the microorganisms at 1.0 to 8.0 days. -1 Or 0.04 to 0.3h -1 .

5. The method according to any one of claims 1 to 4, wherein controlling the input flow comprises maintaining a hydrogen concentration of 0.5 to 20 mg / L, an oxygen concentration of 0.5 to 5 mg / L, and a carbon dioxide concentration of 20 to 2000 mg / L in the liquid phase at a temperature of 28 to 45°C and a gas phase pressure of 100 to 2000 kPa.

6. The method according to any one of claims 1 to 5, wherein the microorganism utilizes hydrogen at a rate of 0.1 to 1.0 mol / g / h, utilizes carbon dioxide at a rate of 0.2 to 0.5 mol / g / h, and utilizes oxygen at a rate of 0.02 to 0.5 mol / g / h.

7. The method according to any one of claims 1 to 6, wherein controlling the input flow comprises maintaining a hydrogen transfer rate of at least 0.2 mol / L / h in the liquid phase and maintaining an oxygen transfer rate of 0.01 to 0.4 mol / L / h in the liquid phase.

8. The method according to any one of claims 1 to 7, wherein controlling the input flow comprises maintaining oxygen in the liquid phase at a transfer rate of 0.015 to 0.25 mol / L / h, preferably 0.03 to 0.1 mol / L / h.

9. The method according to any one of claims 1 to 8, wherein controlling the input flow comprises maintaining a molar ratio of dissolved hydrogen:oxygen:carbon dioxide in the liquid phase of 30 to 60:0.795 to 4.25:0.75 to 2.

0.

10. The method according to any one of claims 1 to 9, wherein controlling the input flow comprises maintaining the molar ratio of hydrogen:oxygen:carbon dioxide dissolved in the liquid phase within a range of 0 to 750 mm from the gas phase, wherein the distance between the gas phase and the liquid phase in direct contact is 12 to 40: 0 to 1.0625: 0.75 to 2.

0.

11. The method according to any one of claims 1 to 10, wherein controlling the input flow comprises maintaining the molar ratio of hydrogen to oxygen in the liquid phase at 0.5:1 to 7:

1.

12. The method according to any one of claims 1 to 11, wherein: (i) Controlling the input flow includes: maintaining hydrogen, oxygen, and carbon dioxide in the liquid phase at a gas transfer coefficient of 10 to 5000 h⁻¹; and / or adding a hydrogen:oxygen molar ratio of 1:1 to 10:1 to the liquid phase; and (ii) Controlling the input flow and nutrient composition includes maintaining the concentration of microorganisms in the liquid phase of the bioreactor at at least 10 g / L, preferably 10 to 100 g / L.

13. Use of biomass obtained by the method of any one of claims 1 to 12 for feeding or providing nutrition to one or more organisms, preferably wherein said organisms include fish, crustaceans, mollusks, poultry, pigs and cattle.

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