Method for producing biomass using hydrogen-oxidizing bacteria

Abstract

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

Description

Technical Field

[0001] The present invention provides methods for producing biomass comprising at least 65% protein by hydrogen-oxidizing microorganisms using one or more input streams comprising one or more gaseous substrates. Background Art

[0002] There is a need to produce protein as a food source in a more sustainable manner to reduce resource use and greenhouse gas (GHG) emissions. The growing global population has put increasing pressure on the availability of resources and the environment. Animal products such as meat, fish, milk and eggs are important sources of protein diets, but livestock use large areas of agricultural land, energy and water. This is mainly due to the fact that animal feed consists of a large number 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 adjusted in composition to suit specific nutritional needs. Due to the efficiency of land, energy and water use in microbial protein production, microbial protein is considered to be a highly sustainable source of protein.

[0003] Many industrial processes generate waste streams containing carbon dioxide and other gaseous components. Examples are energy production by combustion, lime production, fertilizer production, and cement production, which are major sources of atmospheric carbon dioxide and other greenhouse gases (GHGs). Carbon oxides from industrial sources, primarily produced by the combustion of fossil fuels and / or chemicals, are classified as GHGs due to their contribution to adverse environmental conditions. Other industrial processes that involve the combustion of waste include municipal solid waste, sewage sludge, plastics, tires, agricultural residues, and coal- or gas-fired power plants.

[0004] Microorganisms require a carbon source to survive, grow, and produce chemical products. Therefore, carbon oxides derived from industrial gas 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.

[0005] It would therefore be desirable to be able to convert gaseous feedstocks into sources of high quality biomass.There is therefore a need for improved, simple, high yield and economical methods for the biological production of biomass that can be used, for example, as animal feed or for direct human consumption.

[0006] Previous work is 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 (preferably by maintaining a high concentration of microorganisms in the liquid phase of the bioreactor) in combination with high protein content in a continuous and stable manner without making the method economically unattractive is a challenge.

[0007] Patent US9157058B2 (WO2013090769A2) describes apparatus and methods for growing and maintaining microorganisms and / or bioprocesses using one or more gases as electron donors, electron acceptors, carbon sources, or other nutrients, as well as bioprocesses for converting hydrogen and carbon dioxide, or synthesis gas, 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.

[0008] Patent US9206451B2 describes a system and method for capturing carbon from industrial waste using chemoautotrophic microorganisms, but it does not disclose controlled optimal process conditions for growth and maintenance, nor does it disclose preferred microorganisms, nor does it disclose methods for optimizing the protein production capacity of microorganisms.

[0009] Patent application WO2018144965A1 describes microorganisms and bioprocesses for converting gaseous substrates (e.g., renewable hydrogen and waste carbon dioxide producer 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.

[0010] Patent application WO2019010116A1 describes a method for producing polycarbonate compounds from simple gaseous feedstocks such as carbon dioxide, hydrogen, and oxygen by culturing a consortium of microbial cells specifically selected for this purpose in an aqueous 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.

[0011] TG Volova and VA Barashkov in “Characteristics of Proteins Synthesized by Hydrogen-Oxidizing Microorganisms”; Applied Biochemistry and Microbiology, 2010 describe a method in which hydrogen-oxidizing bacteria are cultivated to produce biomass with a dry weight protein content of 64% to 76%, but the amounts of carbon dioxide, hydrogen and oxygen used are not disclosed, nor are methods for optimizing productivity.

[0012] Patent application US2010120104A1 discloses a multi-step process for producing biomass by capturing carbon via obligate and / or facultative chemoautotrophic microorganisms and / or cell extracts containing enzymes from chemoautotrophic microorganisms. A variety of different electron donors and acceptors, as well as microorganisms, are disclosed for use in the process, but specific process parameters are not described.

[0013] Patent application WO2011139804A2 discloses a method for producing biomass by capturing carbon with one carbon atom using hydrogen-oxygen microorganisms and a suitable bioreactor utilizing hydrogen and oxygen, wherein the volume of the gas accounts for at least approximately 2% of the total volume of the column in which it is located. Specific process parameters for optimizing protein production capacity are not described.

[0014] Patent application WO2017165244A1 discloses a method for producing biomass by capturing and converting inorganic and / or organic molecules containing only one carbon atom by chemoautotrophic microorganisms. It is disclosed that Cupriavidus necator is grown in a standard off-the-shelf laboratory-scale bioreactor with the aid of H2 and CO2 to a dry biomass density of more than 40 g / liter within 6 days. It is also disclosed that for samples collected during the arithmetic growth period, 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 more than 70% and more than 80% of total protein by weight, respectively. However, it is disclosed that Cupriavidus necator grows under oxygen-limited conditions, which is not optimal for producing biomass with a high protein concentration at high industrial rates. In addition, the disclosed system for growing Cupriavidus necator is a continuous fed-batch system in which the specific growth rate is a function of an undisclosed gas transfer rate. Thus, WO2017165244A1 does not disclose that biomass can be produced at a high rate, wherein the biomass comprises 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.

[0015] Morinaga et al., "Growth Characteristics and Cell Composition of Alcaligenes eutrophus in Chemostat Culture", Agric. Biol. Chem., 1977, describe conditions for culturing Alcaligenes eutrophus. However, they do not disclose how to achieve biomass production at a rate greater than about 7.2 g / l / day and a protein content greater than 65%, nor do they disclose a growth medium capable of supporting high concentrations of microorganisms. Therefore, combining high productivity with high microbial densities is a challenge.

[0016] Therefore, there remains a need to identify a group of chemoautotrophic microorganisms that can be grown in novel or conventional, controllable and scalable controlled reactor vessels and produce proteins and other nutritionally beneficial products at high productivity in a stable and commercially viable manner. The growth and maintenance of these microorganisms can then be managed by controlling optimal process conditions to fine-tune the metabolic and physiological characteristics of the microorganisms, ultimately resulting in high yields of high-quality biomass with high protein content, preferably by maintaining a high concentration of the microorganisms in the liquid phase of the bioreactor. Summary of the Invention

[0017] The present invention provides a commercially viable method for producing high-quality biomass with a high protein content at high productivity. This is achieved by using an input stream containing 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 while maintaining safe operating conditions within the bioreactor, which can be achieved by mitigating potentially explosive gas mixtures.

[0018] It is therefore an object of the present invention to provide a method for producing biomass by hydrogen-oxidizing microorganisms using one or more input streams comprising one or more gaseous substrates, said biomass comprising at least 65% protein by dry weight of the total biomass, said gaseous substrates comprising hydrogen and / or oxygen and / or carbon dioxide, said method comprising contacting the microorganisms in a liquid phase with a nutrient composition comprising carbon- and / or nitrogen- and / or phosphorus-containing compounds and the gaseous substrates, wherein the input streams and the nutrient composition are controlled and wherein the biomass is produced at a rate of greater than 10 g / l / day.

[0019] Another object is to provide a method wherein controlling the input flow comprises maintaining a molar ratio of dissolved hydrogen:oxygen:carbon dioxide in the liquid phase of 0 to 12.748:0 to 4.25:0 to 2.0 at a distance of 0 mm to 500 mm, preferably 0 mm to 100 mm, from the gas phase in direct contact with the liquid phase.

[0020] Yet another object is to provide a method for producing biomass by bacteria selected from the genus Alcaligines sp.

[0021] Yet another object is to provide a method for producing biomass by bacteria selected from the genus Cupriavidus.

[0022] In another aspect, the present invention provides a method of separating produced biomass and removing nutrient compositions, the method including downstream processing.

[0023] Another object is to provide a method for separating produced biomass by removing a nutrient composition, said method comprising dehydrating and / or drying the biomass such that the biomass comprises a water content of less than 5% by weight.

[0024] Yet another object is to provide a biomass comprising protein, wherein the protein comprises an amino acid content comprising: a histidine content of 0.9% to 4.8% of the total biomass dry weight protein content, an isoleucine content of 2.0% to 6.9% of the total biomass dry weight protein content, a leucine content of 3.8% to 12.0% of the total biomass dry weight protein content, a lysine content of 3.0% to 11.1% of the total biomass dry weight protein content, a methionine content of 1.1% to 5.4% of the total biomass dry weight protein content, a phenylalanine content of 1.7% to 8.5% of the total biomass dry weight protein content, a threonine content of 1.6% to 6.9% of the total biomass dry weight protein content, a tryptophan content of 0.4% to 3.9% of the total biomass dry weight protein content, and a valine content of 1.7% to 9.3% of the total biomass dry weight protein content.

[0025] In another aspect, the present invention provides a biomass comprising a lipid content of 2.3% to 18% of the total biomass dry weight, the lipid content comprising a fatty acid content comprising: a C16:0 palmitic acid content of 23% to 60% of the total biomass dry weight fatty acid content, a C16:1 palmitoleic acid content of 3.8% to 22.3% of the total biomass dry weight fatty acid content, and a C17:1 heptadecenoic acid content of 23% to 60% of the total biomass dry weight fatty acid content.

[0026] Yet another object is to provide the use of the nutrient composition obtained by the method of separating the produced biomass as a nutrient composition for producing biomass.

[0027] It is another object of the present invention to provide the use of biomass, wherein the biomass is used to feed or provide nutrition to one or more organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Preferred embodiments of systems for performing the methods are disclosed.

[0029] Figure 2 Examples of ranges of amounts of essential amino acids per 100 g of total protein content of isolated biomass produced according to the method of the present invention are shown.

[0030] Figure 3 Shown are examples of the proportion of the most abundant fatty acids in the total fatty acid content of isolated biomass produced according to the method of the present invention.

[0031] Figure 4 Examples of ranges of amounts of essential amino acids per 100 g of total protein content of isolated biomass produced according to the method of the invention are shown compared to the ranges of amounts of essential amino acids of typical soybean meal and fish meal used in animal agriculture.

[0032] Figure 5 shows the results of the experiment under different oxygen ( Figure 5A ) and hydrogen ( Figure 5B ) input concentration percentage.

[0033] Figure 6 Predictions of protein content by dry weight of total biomass from hydrogen-oxidizing microorganisms produced according to the present invention are shown, where controlling the input flow includes adding a certain molar ratio of hydrogen:oxygen in the liquid phase.

[0034] Figure 7 Predictions of protein content by dry weight of total biomass from hydrogen-oxidizing microorganisms produced according to the present invention are shown, where controlling the input flow includes controlling the specific growth rate.

[0035] Figure 8A Predictions of preferred hydrogen transfer rates are shown in relation to biomass production rates produced according to the present invention.

[0036] Figure 8B Predictions of preferred oxygen transfer rates are shown in relation to biomass production rates produced according to the present invention.

[0037] The following list includes definitions of the reference numerals as used in the accompanying drawings:

[0038] 1 Oxygen / air input

[0039] 2 Hydrogen input

[0040] 3 Carbon dioxide input

[0041] 4. Inorganic nitrogen (such as urea) addition

[0042] 5 pH buffer addition

[0043] 6 Liquid Growth Medium Addition

[0044] 7 Unused gas recycling

[0045] 8. Removal of liquid containing biomass

[0046] 9 Downstream processing steps

[0047] 10 Liquid Recirculation

[0048] 11 Optional Downstream Processing Steps DETAILED DESCRIPTION

[0049] definition

[0050] Biomass is herein understood to mean the total weight of the microorganisms and their progeny, products and / or metabolites.

[0051] A bioreactor is herein understood to be a system for maintaining and / or growing microorganisms, which comprises a gas phase, usually referred to as the headspace, and a liquid phase. The microorganisms are grown and maintained in the liquid phase.

[0052] Hydrogen-oxidizing microorganisms are understood to include 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 taxa. This group is defined by the ability to use gaseous hydrogen as an electron donor and oxygen as an electron acceptor and to fix carbon dioxide.

[0053] An input stream is understood herein to mean a supply of nutrients and / or energy for the growth and / or maintenance of the microorganisms, including in liquid and / or gaseous phases.

[0054] As used herein, liquid phase is understood to mean a volume containing liquid material. In the liquid phase, microorganisms are typically grown and maintained. Biomass is primarily present within the liquid phase. The liquid phase may also contain solid material that serves as a growth and maintenance substrate for microbial attachment.

[0055] The liquid phase may contain carbon-, nitrogen- and / or phosphorus-containing compounds, wherein the carbon-containing compound may be formate or methanol, but is preferably understood to be essentially limited to dissolved CO 2 or urea, the latter of which may also be considered a source of bioavailable N 2. Formate or methanol can be converted into CO 2 in the liquid phase of the bioreactor, which can be catalyzed, for example, by enzymes present inside or outside the microorganisms, thereby indirectly serving as a supply of gaseous substrate.

[0056] The gas phase is understood herein to mean the volume consisting of gaseous material and in contact with the liquid phase. The gas phase in a bioreactor is often referred to as the headspace and is typically located directly above the liquid phase. For clarity, gases or gaseous substrates that are bubbled into the liquid phase are not part of the gas phase, but become part of the gas phase upon leaving the liquid phase.

[0057] Gaseous substrate is understood to mean a gaseous supply of nutrients and / or energy for the growth and / or maintenance of the microorganisms.

[0058] A cement kiln is understood to mean a 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] Synthesis gas or synthesis gas is understood herein to mean a mixture comprising carbon monoxide, carbon dioxide and hydrogen. Synthesis gas is produced by gasifying carbonaceous fuels into gaseous products. The exact chemical composition of synthesis gas varies based on raw materials and processes. One of the uses of synthesis gas is as a fuel for producing steam or electricity. Another use is as a basic chemical building block for many petrochemical and refining processes. Synthesis gas can be produced from many sources including natural gas, coal, oil-based materials, biomass, other materials that will be discarded as waste, or almost any hydrocarbon feedstock.

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

[0061] Bubbling is understood to mean the process of bubbling gas through a liquid.

[0062] The dry weight or dry matter of a material is understood herein to mean a material consisting of all its components but containing essentially no water. Examples of obtaining the dry weight or dry matter of a material are the use of centrifugation, drum drying, belt drying, evaporation, freeze drying, heating, spray drying, vacuum drying and / or vacuum filtration such that the water content of the material is removed.

[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 killing step process, a dehydration process or a drying process.

[0064] The killing step is understood to mean a process that achieves the reproductive inactivation of microorganisms. This process can take place in the liquid phase, the dehydrated liquid phase or the dried biomass, for example by using ultra-high pressure homogenizers, acids, bases, solvents or heat-based microbial killing methods.

[0065] Dehydration is understood in this article to mean the first process of removing liquid and / or nutrient composition from biomass or a composition comprising biomass. Examples of dehydration are centrifugation, evaporation, heating, tangential flow filtration and vacuum filtration. Dehydration can be followed by other downstream processing.

[0066] Drying is understood to mean the process of removing water from biomass or a composition comprising biomass to produce a biomass consisting of all its components but comprising essentially no water. Examples of drying are drum drying, belt drying, freeze drying, spray drying and vacuum drying.

[0067] Purification of carbon dioxide from exhaust gases originating from production or combustion processes is understood to mean obtaining a volume consisting essentially of carbon dioxide, wherein the other elements of the exhaust gas are substantially removed by means 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 in a post-combustion process by separation methods known in the art, such as by using solvents such as amines to form carbonates. The carbon dioxide is absorbed by the solvent and can then be released by heat to form a highly purified carbon dioxide stream.

[0068] Bioavailable nitrogen is understood to mean all nitrogen species that are readily taken up by microorganisms, including, for example, urea, ammonia and amino acids. For the sake of clarity, it does not include molecular nitrogen (N2).

[0069] Process limiting is understood to mean the situation in which a substance can be measured to zero or close to zero in the liquid phase.

[0070] A chemostat is understood to mean a bioreactor in which the chemical environment is kept in a more or less stable state with respect to, for example, microorganism concentration, pH, (dissolved) gaseous substrates, nutrient composition, liquid phase volume and other parameters known to the person skilled in the art.

[0071] Embodiments of the invention

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

[0073] In some embodiments, the present invention relates to a chemoautotrophic microorganism that can be used for the growth of chemoautotrophic microorganisms for use in human and animal nutrition products and other chemicals. Source includes but is not limited to industrial tail gas, industrial smoke and industrial generated gas, and direct air capture gas and original position electrochemical production gas. Chemoautotrophic metabolism herein refers to the metabolic pattern that microorganisms take in inorganic carbon (for example, by capturing carbon dioxide or formate or methanol as main carbon source) and obtain energy from chemical source (for example, by making hydrogenation). By converting inorganic carbon into organic carbon, these microorganisms serve as primary producers in natural environment. Many of these chemoautotrophic microorganisms can be cultivated in bioreactor with direct or indirect gaseous feedstock for commercial production biomass, and the biomass can be processed into nutritional products, for example, animal feed, companion animal feed or even the human food.

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

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

[0076] The biomass according to the present invention comprises cell masses of microorganisms and / or their products. Preferably, the biomass is a cell mass of microorganisms and / or their products. More preferably, the biomass is a cell mass of microorganisms.

[0077] According to the method of the present invention, most preferably, the one or more gaseous substrates comprise hydrogen, oxygen and carbon dioxide. Preferably, the one or more gaseous substrates comprise hydrogen and oxygen. Preferably, the one or more gaseous substrates comprise hydrogen and carbon dioxide. Preferably, the one or more gaseous substrates comprise oxygen and carbon dioxide. Preferably, the one or more gaseous substrates comprise hydrogen. Preferably, the one or more gaseous substrates comprise oxygen. Preferably, the 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 and a phosphorus-containing compound. Preferably, the nutrient composition comprises a carbon-containing compound. Preferably, the nutrient composition comprises a nitrogen-containing compound. Preferably, the nutrient composition comprises a phosphorus-containing compound.

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

[0080] The 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, the biomass is produced at a rate of 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 10 ... l / 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 5 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, 13.5 g / l / day to 80 g / l / day, 13.5 g / l / day to 50 g / l / day, 14.0 g / l / day to 100 g / l / day, 14.0 g / l / day to 90 g / l / day, 14.0 g / l / day to 80 g / l / day, 14.0 g / l / day to 50 g / l / day, 14.5 g / l / day to 100 g / l / day, 14.5 g / l / day to 90 g / l / day, 14.5 g / l / day to 80 g / l / day, 14.5 g / l / day to 50 g / l / day, 15.0g / l / day to 100g / l / day, 15.0g / l / day to 90g / l / day, 15.0g / l / day to 80g / l / day, 15.0g / l / day to 50g / l / day, 15.5g / l / day to 100g / l / day, 15.5g / l / day to 90g / l / day, 15.5g / l / day to 80g / l / day, 15.5g / l / day to 50g / l / day, 16 .0g / l / day to 100g / l / day, 16.0g / l / day to 90g / l / day, 16.0g / l / day to 80g / l / day, 16.0g / l / day to 50g / l / day, 16.5g / l / day to 100g / l / day, 16.5g / l / day to 90g / l / day, 16.5g / l / day to 80g / l / day, 16.5g / l / day to 50g / l / day, 17 .0g / l / day to 100g / l / day, 17.0g / l / day to 90g / l / day, 17.0g / l / day to 80g / l / day, 17.0g / l / day to 50g / l / day, 18.0g / l / day to 100g / l / day, 18.0g / l / day to 90g / l / day, 18.0g / l / day to 80g / l / day, 18.0g / l / day to 50g / 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] In addition, using highly concentrated gaseous substrate sources has reduced the volume of other non-substrate gases in the microbial environment, thereby improved the efficiency of the gaseous substrate added, and also made it possible to realize the control of the improvement of the amount of gaseous substrate in the microbial environment. Gaseous substrate can be added in the liquid phase separately or as any premixed combination. According to the present invention, preferably the gaseous substrate is added with non-substrate gases such as nitrogen or carbon monoxide at low concentration as far as possible. One or more gaseous substrates are added in the liquid phase and comprise contacting the gaseous substrate with the liquid phase so that the gaseous substrate mixes with at least a portion of the liquid phase. When a certain proportion of one or more gaseous substrates are added in the liquid phase, those skilled in the art will appreciate 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 9 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] The use of a highly concentrated gaseous substrate source reduces the volume of other non-substrate gases in the microbial environment, ideally resulting in a gas phase consisting only of hydrogen, oxygen, and carbon dioxide.

[0084] Thus, also provided herein is a process of the invention, wherein the gas phase consists essentially of only hydrogen, oxygen and carbon dioxide.

[0085] Preferably, the input flows are controlled by adding hydrogen at a concentration of 10% to 100% (v / v), oxygen at a concentration of 2% to 100% (v / v), and carbon dioxide at a concentration of 2% to 100% (v / v), either alone or in any premixed combination thereof, to the liquid phase. More preferably, the input flows are controlled by adding hydrogen at a concentration of 80% to 100% (v / v), oxygen at a concentration of 20% to 100% (v / v), and carbon dioxide at a concentration of 5% to 100% (v / v), either alone or in any premixed combination thereof, to the liquid phase. More preferably, the input flow is controlled by adding to the liquid phase, alone or as any premixed combination thereof, a concentration 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 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) of hydrogen,The concentration 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), 2% to 90% (v / v), 5% to 90% (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) 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) , 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) carbon dioxide. Even more preferably, the input flows are controlled by adding hydrogen at a concentration of 70% to 100% (v / v), oxygen at a concentration of 20% to 100% (v / v), and carbon dioxide at a concentration of 5% to 100% (v / v) to the liquid phase, either alone or as any premixed combination thereof.

[0086] The substrate gas is preferably supplemented with as low a concentration as possible of a non-substrate gas such as nitrogen or carbon monoxide.

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

[0088] Preferably, carbon dioxide originating from waste gases of production or combustion processes 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), 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). % (v / v), 0% 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 mainly found in many bacteria, including but not limited to purple non-sulfur bacteria such as Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodpsuedamonas palustris, Pseudomonas such as Pseudomonas carboxydovorans, water-producing bacteria such as Hydrogenobacter thermophilus, methanogens such as Methanobacterium thermoautotrophicum, α-Proteobacteria such as Xanthobacter flavus, β-Proteobacteria such as Ralstonia metallidurans, Cupribotium nektonii, γ-Proteobacteria such as Hydrogenovibrio marinus, ε-Proteobacteria such as Helicobacter pylori, and β-Proteobacteria such as Xanthobacter flavus. pylori), acetogenic bacteria such as Acetobacterium woodii, or other microorganisms expressing hydrogen uptake and carbon dioxide fixation metabolism, whether endogenous or introduced by genetic manipulation, mutation, selection or directed evolution. Most of these microorganisms are capable of heterotrophic metabolism as well as phototrophic metabolism, or a mixed metabolism using both energy and carbon sources. Hydrogen is used as the energy source, and carbon dioxide is used as the carbon source. Carbon monoxide can also serve as both the energy source and the carbon source.

[0090] Preferably, the microorganisms according to the method of the present invention comprise bacteria selected from the group consisting of Rhodopseudomonas sp., Rhodospirillum sp., Rhodococcus sp., Rhodobacter sp., Rhizobium sp., Thiocaps sp., Pseudomonas sp., Nocardia sp., Hydrogenomas sp., Hydrogenobacter sp., Hydrogenovibrio sp., Helicobacter sp., Xanthobacter sp., Hydrogenophaga sp., Bradyrhizobium sp., Ralstonia sp. sp.), Gordonia sp., Mycobacteria sp., Alcaligenes sp., Cupriavidus sp., Variovorax sp., Acidovorax sp., Anabaena sp., Scenedesmus sp., Chlamydomonas sp., Ankistrodesmus sp., Rhaphidium sp., or Arthrobacter sp., and combinations thereof. More preferably, the microorganism comprises a bacterium selected from the genus Alcaligenes or Cupriavidus. More preferably, the microorganism comprises a bacterium selected from the genus Alcaligenes. More preferably, the microorganism comprises a bacterium selected from the genus Cupriavidus. Even more preferably, the microorganism comprises a bacterium selected from the species Cupriavidus.

[0091] 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 for nutritional quality.

[0092] The ratio of protein, lipid, DNA, RNA and other components of cell biomass is mediated by growth conditions, growth rate and carbon-nitrogen ratio. Therefore, the control of substrate availability and general process conditions directly affects the amount and quality of biomass produced during the fermentation process. Maximum baseline productivity needs to avoid substrate limitation, but as the density of microorganisms in fermentation increases, the substrate utilization rate exceeds the supply, particularly in the case of gaseous substrates. This means that high gas flow is required to establish and maximize productivity. However, it is challenging to achieve this in the case of explosive gas mixtures comprising hydrogen and oxygen. When combined with a ratio comprising more than 5% oxygen and more than 4% hydrogen, gas mixtures containing hydrogen and oxygen produce explosive mixtures, as disclosed in, for example, 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 the initial addition of gases to the system above the explosion limit as they are initially injected into the liquid culture medium phase. Feedback control then ensures that the system fully utilizes oxygen and / or hydrogen so that the gases contributing to the headspace can be kept below the explosion safety limit, which under standard conditions is 5% (v / v) for oxygen and 4% (v / v) for hydrogen. Furthermore, all three gas inputs are controlled so that either 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 regulation of biomass composition by direct metabolic restrictions and physiological restrictions imposed on the microorganism. Ammonium hydroxide and / or other biologically available nitrogen sources are supplied to the process directly and / or by being incorporated into the liquid culture medium input stream and / or the recirculating liquid stream so as to provide a minimum of 10 g of atomic nitrogen for every 100 g of produced biomass, controlled as a part of a feedback loop in response to the density and dilution rate of the cells in the liquid phase. In addition to gaseous substrates, hydrogen-oxidizing bacteria also require a biologically available nitrogen source for protein production. This nitrogen is the main contributing factor to protein content and has nothing to do with the respective molecular association in, for example, ammonium hydroxide (NH4OH) or ammonium chloride (NH3Cl). Since excessive biologically available nitrogen in the liquid phase will be recycled, a maximum threshold value is not foreseen.

[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 nutrient composition is controlled to include the addition of 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 nutrient composition is controlled to include the addition of 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 control nutrient composition comprises adding 10.5 g to 50000 g, 11.0 g to 50000 g, 11.5 g to 50000 g, 12.0 g to 50000 g, 12.5 g to 50000 g, 13.0 g to 50000 g, 13.5 g to 50000 g, 14.0 g to 50000 g, 14.5 g to 50000 g, 15.0 g to 50000 g, 16 g to 50000 g, 17 g to 50000 g, 18 g to 50000 g, 19 g to 50000 g, 20 g to 50000 g, 21 g to 50000 g, 23 g to 50000 g, 24 g to 50000 g, 25 g to 50000 g, 26 g to 50000 g, 27 g to 50000 g, 28 g to 50000 g, 29 g to 50000 g, 30 g to 50000 g, 31 g to 50000 g, 32 g to 50000 g, 33 g to 50000 g, 34 g to 50000 g, 35 g to 50000 g, 36 g to 50000 g, 37 g to 50000 g, 38 g to 50000 g, 39 g to 50000 g, 40 g to 50000 g, 41 g to 50000 g, 42 g to 50000 g, 43 g to 50000 g 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, 110 g to 50000g, 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 g to 50,000 g, 20,000 g to 50,000 g, 25,000 g to 50,000 g, 30,000 g to 50,000 g, 35,000 g to 50,000 g, 40,000 g to 50,000 g, 45,000 g to 50,000 g, 50,000 g to 50,000 g, 60,000 g to 50,000 g, 70,000 g to 50,000 g, 80,000 g to 50,000 g, 90,000 g to 50,000 g, 10,000 g to 50,000 g, 15,000 g to 50,000 g, 20,000 g to 50,000 g, 25,000 g to 50,000 g, or 30,000 g to 50,000 g bioavailable nitrogen.

[0096] According to the present invention, controlling the nutrient composition preferably comprises 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 present invention, controlling the nutrient composition preferably includes adding a growth medium having a pH of 1.0 to 3.0 or 4.0 prior to addition to the liquid phase. Preferably, the pH of the growth medium is 2.2 to 3.2, or about 2.8. The low pH prevents precipitation of components of the growth medium during preparation. The components of the growth medium include those disclosed in Example 1 or substantially similar components 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% volume per hour to maximize production rate, protein content, and quality of the biomass.

[0099] Certain specific replacement rates of the liquid phase within the bioreactor result in higher levels of protein content and quality of the biomass. Preferably, the liquid phase of the bioreactor in which the microorganisms are grown and maintained is replaced per hour at the following volume: 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 % 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 rate at which the gas dissolves in the liquid phase and its distribution throughout the liquid phase. The volumetric mass transfer coefficient (kLa) represents the rate or efficiency with which a concentration gradient of a dissolved gas is transferred from its gas phase to the liquid phase. In order to obtain high utilization of the gaseous substrate and subsequent high biomass and protein production rates, it is necessary to maintain as high a kLa as possible. Preferably, the gaseous substrate is added to the liquid phase at as high a concentration as possible to increase the driving force for dissolution and diffusion of the gas into the bulk liquid phase. Preferably, the gaseous substrate is added at its saturation concentration.

[0100] Gaseous substrate ratios, concentrations, and microbial consumption need to be maintained by adequate gas transfer into the liquid phase. Controlling the input flow comprises maintaining a gas transfer coefficient of hydrogen, oxygen, and carbon dioxide in the liquid phase of 1 / hour to 5000 / hour. Preferably, controlling the input flow comprises maintaining a gas transfer coefficient of hydrogen, oxygen, and carbon dioxide in the liquid phase of 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. hours, 10 / hour to 3000 / hour, 20 / hour to 3000 / hour, 50 / hour to 3000 / hour, 100 / hour to 3000 / hour, 200 / hour to 3000 / hour, 400 / hour to 3000 / hour, 600 / hour to 3000 / hour, 800 / hour to 3000 / hour, 1000 / hour to 3000 / hour, 1500 / hour to 3000 / hour, 2000 / hour to 3000 / hour, 1 / hour to 2000 / hour, 10 / hour to 2000 / hour, 20 / hour to 2000 / hour, 50 / hour to 2000 / hour, 100 / hour 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, 20 / hour to 1500 / hour, 50 / hour to 1500 / hour, 100 / hour to 1500 / hour, 200 / hour to 1500 / hour, 400 / hour to 1500 / hour, 600 / hour to 1500 / hour, 800 / hour to 1500 / hour hour, 1000 / hour to 1500 / hour, 1 / hour to 1000 / hour, 10 / hour to 1000 / hour, 20 / hour to 1000 / hour, 50 / hour to 1000 / hour, 100 / hour to 1000 / hour, 200 / hour to 1000 / hour, 400 / hour to 1000 / hour, 600 / hour to 1000 / hour, 800 / hour to 1000 / hour, 1 / hour to 800 / hour, 10 / hour to 800 / hour, 20 / hour to 800 / hour, 50 / hour to 800 / hour, 100 / hour to 800 / hour, 200 / hour to 800 / hour,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 the gaseous substrate input result in higher levels of protein content and quality of the biomass. Preferably, controlling the input flow comprises adding hydrogen:oxygen:carbon dioxide in a molar ratio of 2 to 80:0.25 to 20:0.25 to 20 in the liquid phase.More preferably, controlling the input flow comprises adding in the liquid phase a molar ratio of hydrogen:oxygen:carbon dioxide of 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 to 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 to 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 to 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 .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 limiting, and this can be controlled using a range of possible methods, including for example:

[0105] - using a dissolved carbon dioxide probe to monitor dissolved carbon dioxide and using the resulting data as part of a feedback loop to maintain a concentration of about 1 mmol / l or higher by controlling the rate of addition of the input gas; or

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

[0107] With respect to adding a certain molar ratio of gaseous substrate, it was found that controlling the input flow according to the present invention preferably comprises adding hydrogen:oxygen in a molar ratio of 0.5:1 to 12:1 in the liquid phase. More preferably, controlling the input flow comprises adding hydrogen:oxygen in a molar ratio of 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, 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 comprises adding in the liquid phase a molar ratio of hydrogen to oxygen 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 comprises adding in the liquid phase a molar ratio of hydrogen to oxygen 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 hold-up" 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 fully closed bioreactor systems and in bioreactor systems employing gas phase and / or gas-liquid phase recirculation, the gas hold-up composition ratio is preferably maintained by matching the gas input ratio to the gas usage ratio. In open systems, the desired gas hold-up composition is preferably controlled by using that composition, or a very similar composition, as the average input gas composition. Thus, with respect to maintaining a certain molar ratio of gaseous substrates, it has been found that controlling the input flow according to the present invention preferably includes maintaining a molar ratio of hydrogen to oxygen gas hold-up in the liquid phase of 0.5:1 to 7:1. More preferably, controlling the input flow comprises maintaining a molar ratio of hydrogen:oxygen gas hold-up in the liquid phase of 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 comprises maintaining a molar ratio of hydrogen to oxygen gas hold-up in the liquid phase of 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 comprises maintaining a molar ratio of hydrogen to oxygen gas hold-up in the liquid phase of 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] In order to obtain a biomass with a high protein content at a high productivity according to the present invention, it was found that oxygen as a gaseous substrate is preferably added to the liquid phase in the highest possible concentration, taking into account other parameters such as the hydrogen to oxygen ratio. Preferably, according to the present invention, the addition of a hydrogen to oxygen molar ratio to the liquid phase comprises adding oxygen to the liquid phase at a concentration of 5% to 100% (v / v). More preferably, adding a molar ratio of hydrogen to oxygen in the liquid phase according to the present invention comprises adding 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) oxygen to the liquid phase, or 5% to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% (v / v) oxygen to the liquid phase. Even more preferably, adding a molar ratio of hydrogen to oxygen in the liquid phase according to the present invention comprises adding a concentration of 10% to 100% (v / v) oxygen to the liquid phase. Still even more preferably, adding a molar ratio of hydrogen:oxygen to the liquid phase according to the present invention comprises 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 a molar ratio of dissolved hydrogen, oxygen, and carbon dioxide is important for optimal production of biomass. Preferably, controlling the input flow comprises maintaining a molar ratio of dissolved hydrogen:oxygen:carbon dioxide in the liquid phase of 1 to 60:0.5 to 20:0.5 to 20.More preferably, controlling the input flow comprises maintaining a molar ratio of dissolved hydrogen:oxygen:carbon dioxide in the liquid phase of 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, 4 to 20:0.795 to 4.25:0.75 to 2.0, 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 to 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 12.748:0.795 to 4.25:0.5 to 20 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 flows includes maintaining a hydrogen concentration in the liquid phase of 0.5 mg / l to 20 mg / l, an oxygen concentration of 0.5 mg / l to 80 mg / l, and a carbon dioxide concentration of 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 comprises 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, respectively. 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 15mg / l, 0.5mg / l to 80mg / l and 20mg / l to 2000mg / l; 0.5mg / l to 10mg / l, 0.5mg / l to 80mg / l and 20mg / l to 2000mg / l; 2mg / l to 10mg / l, 0.5mg / l to 80mg / l and 20mg / l to 2000mg / l; 5mg / l to 10mg / l, 0.5mg / l to 80mg / l and 20mg / l to 2000mg / l; 0.5mg / l to 5mg / l, 0.5mg / l to 80mg / l and 20mg / l to 2000mg / l. g / l and 20mg / l to 2000mg / l; 1.0mg / l to 5mg / l, 0.5mg / l to 80mg / l and 20mg / l to 2000mg / l; 2.0mg / l to 5mg / l, 0.5mg / l to 80mg / l and 20mg / l to 2000mg / l; 0.5mg / l to 3mg / l, 0.5mg / l to 80mg / l and 20mg / l to 2000mg / l; 1mg / l to 3mg / l, 0.5mg / l to 80mg / l and 20mg / l to 2000mg / l;

[0114] 0.5mg / l to 20mg / l, 2mg / l to 80mg / l and 20mg / l to 2000mg / l; 0.5mg / l to 20mg / l, 5mg / l to 80mg / l and 20mg / l to 2000mg / l; 0.5mg / l to 20mg / l, 10mg / l to 80mg / l and 20mg / l to 2000mg / l; 0.5mg / l to 20mg / l, 20mg / l to 80mg / l and 20mg / l to 2000mg / l; 0.5mg / l to 20mg / l, 40mg / l to 80mg / l and 20mg / 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 50mg / l and 20mg / l to 2000mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 20mg / l and 20mg / l to 2000mg / l; 0.5mg / l to 20mg / l, 2mg / l to 20mg / l and 20mg / l to 2000mg / l; 0.5mg / l to 20mg / l, 5mg / l to 20mg / l and 20mg / l to 2000mg / l; 0.5mg / l to 20mg / l, 10mg / l to 20mg / l and 20mg / l to 2000mg / l; 0.5mg / l to 20mg / 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] 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 50mg / l to 2000mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 100mg / l to 2000mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 200mg / l to 2000mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 500mg / l to 2000mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 1000mg / l to 2000mg / l; 0.5mg / l to 20mg / l l, 0.5mg / l to 80mg / l and 20mg / l to 1500mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 50mg / l to 1500mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 100mg / l to 1500mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 200mg / l to 1500mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 500mg / l to 1500mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and l and 1000mg / l to 1500mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 20mg / l to 1000mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 50mg / l to 1000mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 100mg / l to 1000mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 200mg / l to 1000mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 500mg / l to 1000mg / l mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 20mg / l to 500mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 50mg / l to 500mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 100mg / l to 500mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 200mg / l to 500mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 20mg / l to 250mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 50mg / l to 250mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 100mg / l to 250mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 20mg / l to 100mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 50mg / l to 100mg / l; 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 20mg / l to 50mg / l; or 0.5mg / l to 20mg / l, 0.5mg / l to 80mg / l and 30mg / l to 50mg / l.

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

[0117] It has been found that maintaining a minimum transfer rate of hydrogen and / or oxygen in the liquid phase is necessary to produce biomass comprising at least 65% protein at a rate of at least 10 g / l / day, and it is further preferred to maintain a concentration of microorganisms in the liquid phase of the bioreactor of at least 10 g / l. Considering only biomass production rate and quality, theoretically no maximum transfer rate is required, however commercial, cost and safety considerations dictate the imposition of a maximum transfer rate.

[0118] Preferably, according to the present invention, controlling the input flow comprises maintaining a transfer rate of hydrogen in the liquid phase of at least 0.02 mol / l / hour and / or a transfer rate of oxygen in the liquid phase of at least 0.003 mol / l / hour. More preferably, controlling the input flow comprises maintaining a transfer rate of hydrogen in the liquid phase of 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, 0.13 mol / l / hour. hour, 0.14mol / l / hour, 0.15mol / l / hour, 0.16mol / l / hour, 0.17mol / l / hour, 0.18mol / l / hour, 0.19mol / l / hour, 0.2mol / l / hour, 0.21mol / l / hour, 0.22mol / l / hour, 0.23mol / l / hour, 0.24mol / l / hour, 0.25mol / l / hour, 0.3mol / l / hour, 0.35 mol / l / hour, 0.4 mol / l / hour or 0.5 mol / l / hour, and / or the transfer rate of oxygen 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, 0.06 mol / l / hour, 0.08 mol / l / hour, 0.10 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 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 comprises maintaining a transfer rate of hydrogen in the liquid phase of at least 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, 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 and / or or the transfer rate of oxygen 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 comprises maintaining a transfer rate of hydrogen in the liquid phase of 0.02 mol / l / hour to 3.0 mol / l / hour and / or a transfer rate of oxygen in the liquid phase of 0.01 mol / l / hour to 0.4 mol / l / hour. More preferably, controlling the input flow comprises maintaining a transfer rate of hydrogen in the liquid phase of 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, 0.17 mol / l / hour 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, 0.9 mol / l / hour, 1.0 mol / l / hour, 1.1 mol / l / hour, 1.2 mol / l / hour, 1.3 mol / l / hour, 1.4 mol / l / hour, 1.5 mol / l / hour, 2.0 mol / l / hour or 2.5 mol / l / hour and / or the transfer rate of oxygen in the liquid phase is 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, 0.06 mol / l / hour, 0.08 mol / l / hour, 0.10 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 From 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 to 0.25 mol / l / hour, 0.3 mol / l / hour or 0.35 mol / l / hour.Even more preferably, controlling the input flow comprises maintaining a transfer rate of hydrogen in the liquid phase of from 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, 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, / hour, 0.55 mol / l / hour, 0.6 mol / l / hour or 0.65 mol / l / hour and / or the transfer rate of oxygen in the liquid phase is from 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. In addition, more preferably, the transfer rate of oxygen 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 microorganism utilizes hydrogen, oxygen and carbon dioxide at a rate of 0.03 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, respectively. More preferably, the microorganism utilizes hydrogen, oxygen and carbon dioxide at the following rates: 0.05 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, respectively; 0.1 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; 0.2 mol / g / hour to 1.0 mol / g / hour 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; 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. Hour to 0.5mol / g / hour; 0.05mol / g / hour to 0.5mol / g / hour, 0.01mol / g / hour to 0.5mol / g / hour and 0.01mol / g / hour to 0.5mol / g / hour; 0.1mol / g / hour to 0.5mol / g / hour, 0.01mol / g / hour to 0.5mol / g / hour and 0.01mol / g / hour to 0.5mol / g / hour; 0.2mol / g / hour to 0.5mol / g / hour, 0.01mol 1 / g / hour to 0.5mol / g / hour and 0.01mol / g / hour to 0.5mol / g / hour; 0.03mol / g / hour to 0.2mol / g / hour, 0.01mol / g / hour to 0.5mol / g / hour and 0.01mol / g / hour to 0.5mol / g / hour; 0.05mol / g / hour to 0.2mol / g / hour, 0.01mol / g / hour to 0.5mol / g / hour and 0.01mol / g / hour to 0.5mol / g / hour;

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

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

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

[0124] 0.05mol / g / hour to 1.0mol / g / hour, 0.2mol / g / hour to 0.5mol / g / hour and 0.02mol / g / hour to 0.5mol / g / hour; 0.1mol / g / hour to 1.0mol / g / hour, 0.2mol / g / hour to 0.5mol / g / hour and 0.02mol / g / hour to 0.5mol / g / hour; 0.15mol / g / hour to 1.0mol / g / hour, 0.2mol / g / hour to 0.5mol / g / hour and 0.02mol / g / hour to 0.5mol / g / hour; g / hour, 0.2mol / g / hour to 0.5mol / g / hour and 0.02mol / g / hour to 0.5mol / g / hour; 0.05mol / g / hour to 0.5mol / g / hour, 0.01mol / g / hour to 0.2mol / g / hour and 0.02mol / g / hour to 0.5mol / g / hour; 0.1mol / g / hour to 0.5mol / g / hour, 0.03mol / g / hour to 0. 0.2mol / g / hour and 0.02mol / g / hour to 0.5mol / g / hour; 0.15mol / g / hour to 0.5mol / g / hour, 0.05mol / g / hour to 0.2mol / g / hour and 0.02mol / g / hour to 0.5mol / g / hour; 0.05mol / g / hour to 0.3mol / g / hour, 0.01mol / g / hour to 0.1mol / g / hour and 0.02mol 1 mol / g / hour to 0.5 mol / g / hour; 0.1 mol / g / hour to 0.3 mol / g / hour, 0.03 mol / g / hour to 0.1 mol / g / hour and 0.02 mol / g / hour to 0.5 mol / g / hour; 0.15 mol / g / hour to 0.3 mol / g / hour, 0.05 mol / g / hour to 0.1 mol / g / hour and 0.02 mol / g / hour to 0.5 mol / g / hour;

[0125] 0.05mol / g / hour to 1.0mol / g / hour, 0.03mol / g / hour to 0.5mol / g / hour and 0.2mol / g / hour to 0.5mol / g / hour; 0.1mol / g / hour to 1.0mol / g / hour, 0.03mol / g / hour to 0.5mol / g / hour and 0.2mol / g / hour to 0.5mol / g / hour; 0.15mol / g / hour to 1.0mol / g / hour, 0.03mol / g / hour to 0.5mol / g / hour and 0.2mol / g / hour to 0.5mol / g / hour; g / hour, 0.03mol / g / hour to 0.5mol / g / hour and 0.2mol / g / hour to 0.5mol / g / hour; 0.05mol / g / hour to 0.5mol / g / hour, 0.03mol / g / hour to 0.5mol / g / hour and 0.01mol / g / hour to 0.2mol / g / hour; 0.1mol / g / hour to 0.5mol / g / hour, 0.03mol / g / hour to 0.5mol / g / hour and 0.01mol / g / hour to 0.2mol / g / hour; 0.5mol / g / hour and 0.03mol / g / hour to 0.2mol / g / hour; 0.15mol / g / hour to 0.5mol / g / hour, 0.03mol / g / hour to 0.5mol / g / hour and 0.05mol / g / hour to 0.2mol / g / hour; 0.05mol / g / hour to 0.3mol / g / hour, 0.03mol / g / hour to 0.5mol / g / hour and 0.01mol 1 mol / g / hour to 0.1 mol / g / hour; 0.1 mol / g / hour to 0.3 mol / g / hour, 0.03 mol / g / hour to 0.5 mol / g / hour and 0.03 mol / g / hour to 0.1 mol / g / hour; 0.15 mol / g / hour to 0.3 mol / g / hour, 0.03 mol / g / hour to 0.5 mol / g / hour and 0.02 mol / g / hour to 0.1 mol / g / hour;

[0126] 0.1mol / g / hour to 1.0mol / g / hour, 0.05mol / g / hour to 0.5mol / g / hour and 0.05mol / g / hour to 0.5mol / g / hour; 0.1mol / g / hour to 1.0mol / g / hour, 0.1mol / g / hour to 0.5mol / g / hour and 0.1mol / g / hour to 0.5mol / g / hour; 0.4mol / g / hour to 1.0mol / g / hour, 0.1mol / g / hour to 0.3mol / g / hour and 0.1mol / g / hour to 0.3mol / g / hour; 0.05mol / g / hour to 0.5mol / g / hour, 0.0 3mol / g / hour to 0.15mol / g / hour, 0.02mol / g / hour to 0.1mol / g / hour; 0.1mol / g / hour to 1.0mol / g / hour, 0.08mol / g / hour to 0.36mol / g / hour and 0.06mol / g / hour to 0.26mol / g / hour; 0.08mol / g / hour to 0.75mol / g / hour, 0.06mol / g / hour to 0.27mol / g / hour and 0.05mol / g / hour to 0.2mol / g / hour; 0.08mol / g / hour to 0.5mol / g / hour, 0.06mol / g / hour to 0.18mol / 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 Hour to 0.15 mol / 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 bubbled in the bioreactor from the bottom usually.The microorganism that is present in the bioreactor utilizes gaseous substrate to produce biomass.In this method, oxygen and hydrogen are input as the part of feedback loop and controlled, to allow gas to be initially added to the system higher than explosion limit, because it is initially injected into the liquid culture medium phase.Then, feedback control guarantees that the system makes full use of oxygen and / or hydrogen, makes the gas that causes head space to remain below explosion safety limit, under standard conditions, is 5% (v / v) and is 4% (v / v) explosion safety limit for hydrogen for oxygen.In addition, whole three kinds of gas input are controlled to make oxygen or hydrogen remain the restricted gas in the system.

[0128] Controlling the input flow includes maintaining a molar ratio of dissolved hydrogen:oxygen:carbon dioxide in the liquid phase of 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 within a distance of 0 mm to 750 mm from the gas phase in direct contact with the liquid phase.

[0129] Preferably, controlling the input flow comprises maintaining a molar ratio of dissolved hydrogen:oxygen:carbon dioxide in the liquid phase of 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 comprises maintaining a molar ratio of dissolved hydrogen:oxygen:carbon dioxide in the liquid phase of 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 3.187:0.75 to 6:0.75 to 2.0, 0 to 3.187:0.75 to 2.0, 0 to 3.187:0.25 to 6:0.75 to 2.0, 0 to 3.187:0.75 to 2.0, 0 to 3.187:0.75 to 6:0.75 to 2.0, 0 to 3.187:0.75 to 2.0, 0 to 3.187:0.25 to 4.25:0.75 to 2.0. 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: 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 3.187 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 3.187:0.795 to 4.25:0.5 to 15. .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 comprises maintaining a molar ratio of hydrogen dissolved in the liquid phase to oxygen dissolved in the liquid phase to 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 of 3.183 to 12.748: 0.795 to 4.25: 0 to 0.5,

[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 to 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, 3.183 to 12.748: 2.5 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: 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 .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 comprises maintaining a molar ratio of dissolved hydrogen:oxygen:carbon dioxide 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, the distance comprises 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 by using a bioreactor suitable for use in an industrial environment.Preferably, the bioreactor according to the invention is a chemostat.

[0138] Bioreactors suitable for industrial environments follow standard requirements in the field of industrial fermentation of microorganisms, preferably hydrogen-oxidizing microorganisms. Bioreactors suitable for industrial environments typically have a size of about 0.2 m 3 About 10m 3 The liquid phase volume and the plant-scale bioreactor has a volume of about 2m 3 About 500m 3 The liquid phase volume is 100,000, but there is no theoretical upper limit. 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 present invention include design considerations to prevent and / or withstand the consequences of potentially explosive gas mixtures containing hydrogen and oxygen. Therefore, the bioreactor suitable for industrial environments according to the present invention enables the use of high concentrations of gaseous substrates according to the present invention, such as hydrogen and / or oxygen.

[0139] In the context of the present invention, bioreactor, preferably chemostat, can be used to keep the physiological state and specific growth rate of microorganisms constant.This is achieved by following: keep various continuous (bio) chemical processes such as by controlling stirring speed, gaseous substrate transfer rate, dilution rate (the volume flow of the nutrients supplied divided by total volume), temperature, pH, remove the culture medium that comprises microorganism and / or add culture medium to preferably keep constant liquid culture medium volume basically.For example, by changing the speed that adds culture medium in bioreactor, the specific growth rate of microorganism can be controlled.Increasing dilution rate will increase the growth of microorganism.However, need to control dilution rate with respect to specific growth rate to prevent washing out (wash-out).Controlling dilution rate is in order to maximize protein production rate and protein content of microorganism.When the specific growth rate of microorganism was too high, protein content and / or the quality of biomass may reduce.

[0140] The bioreactor for carrying out the method according to the invention is preferably selected from the group consisting of a bubble column reactor, an airlift reactor, a continuous stirred tank reactor and a circulation reactor. A preferred bioreactor for carrying out the method according to the invention is a continuous stirred tank reactor. Preferably, the bioreactor comprises one or more gas and / or liquid circulation systems.

[0141] The principle behind the higher level of protein content and the quality that changes liquid phase and causes biomass in bioreactor is to keep microorganism in physiological stable state under constant environmental condition.Under this stable state, growth is carried out with constant specific growth rate basically, and all culture parameters such as pH, nutrient concentration, gaseous substrate concentration, microorganism concentration all keep constant basically.Find that such physiological stable state of microorganism is necessary for comprising the biomass of at least 65% protein with the rate production greater than 10g / l / day.Therefore, according to the present invention, preferably control inflow and nutrient composition to keep microorganism specific growth rate and / or stable state, preferred microbial stable state, more preferably microbial specific growth rate.

[0142] According to the present invention, the input flow and nutrient composition are preferably controlled to achieve or maintain a steady state of microorganisms by maintaining a microorganism concentration in the liquid phase of the bioreactor of at least 5 g / l, preferably 5 g / l to 100 g / l. More preferably, the steady state of microorganisms can be achieved or maintained by maintaining a microorganism concentration in the liquid phase of the bioreactor of 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 ... , 8g / l, 9g / l, 10g / l to 11g / l, 12g / l, 13g / l, 14g / l, 15g / l, 16g / l, 17g / l, 18g / l, 19g / l, 20g / l, 25g / l or 30g / l to 50g / l, 60g / l, 70g / l, 80g / l, 90g / l or 100g / l to achieve or maintain a stable state of the microorganisms. Even more preferably, a steady state of microorganisms may be achieved or maintained by maintaining a concentration of the microorganisms in the liquid phase of the bioreactor of 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 comprises maintaining the specific growth rate of the microorganisms 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, controlling the input flow and nutrient composition according to the present invention comprises maintaining the specific growth rate of the microorganisms 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 comprises maintaining the specific growth rate of the microorganism 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, controlling the input flow and nutrient composition according to the present invention comprises maintaining a specific growth rate of the microorganism 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 the microorganism is too high, the protein content and / or quality of the biomass may decrease. For example, the nucleic acid content of the biomass may become too high.

[0145] Thus, preferably, controlling the input flow and the nutrient composition according to the present invention comprises maintaining the specific growth rate of the microorganisms between 1.0 / day and 8.0 / day. More preferably, controlling the input flow and the nutrient composition comprises maintaining the specific growth rate of the microorganisms between 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 and 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 the nutrient composition comprises maintaining the specific growth rate of the 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 the nutrient composition according to the present invention comprises maintaining the specific growth rate of the microorganisms at 0.03 / hour to 0.4 / hour. More preferably, controlling the input flow and nutrient composition comprises maintaining the specific growth rate of the microorganism from 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 comprises maintaining the specific growth rate of the microorganism from 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 found that bacteria selected from the genus Alcaligenes are capable of producing high-quality biomass at a high production rate. Therefore, the present invention relates to a method for producing biomass by bacteria selected from the genus Alcaligenes. The applicant has unexpectedly found that bacteria selected from the genus Cuprilobacter are capable of producing even higher-quality biomass at an even higher production rate. Therefore, the present invention relates to a method for producing biomass by bacteria selected from the genus Cuprilobacter, a preferred species of Cuprilobacter. Preferably, the biomass comprises at least 65% protein of the total biomass by dry weight. Preferably, the biomass is produced at a rate greater than 10 g / l / day. Preferably, the method for producing biomass comprises using one or more input streams comprising one or more gaseous substrates, the gaseous substrate comprising hydrogen, oxygen and / or carbon dioxide, the method comprising contacting the microorganisms in the liquid phase with a nutrient composition comprising a compound containing carbon, nitrogen and / or phosphorus and the gaseous substrate, wherein the input stream and the nutrient composition are controlled. The present invention also relates to a method for producing biomass by bacteria selected from the genus Alcaligenes, separating the produced biomass and removing the nutrient composition, the method comprising downstream processing. The present invention also relates to a method for producing biomass by bacteria selected from the genus Cupriatus, preferably Cupriatus necator, separating the produced biomass and removing the nutrient composition, said method including downstream processing.

[0147] In order that the biomass produced is further applied as for example the nutrient source that is used for other organisms, it is necessary to process the biomass produced.In the agricultural food industry, usually remove the nutrient composition and the water component of the biomass produced.For the convenience of transportation, storage and prevent being polluted by pathogen or other undesirable organisms, the water content of the biomass produced that is used for further application needs to be as low as possible.Therefore, the invention still further relates to the method for separating the biomass produced according to the method for producing according to the invention and removing the nutrient composition, and said method comprises downstream processing.In addition, the present invention relates to the method for separating the biomass produced according to the invention and removing the nutrient composition, and said method comprises that biomass is dehydrated and / or dried so that biomass comprises the water content less than 10 weight %. Preferably, the biomass comprises a water content of less than 9.0%, 8.0%, 7.0%, 6.5%, 6.0%, 5.5%, 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0% or 0.5% by weight.

[0148] The nutrient composition obtained by separating the biomass produced according to the method of the present invention and removing the nutrient composition (including downstream processing) or by separating the 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 weight%, 9.0 weight%, 8.0 weight%, 7.0 weight%, 6.5 weight%, 6.0 weight%, 5.5 weight%, 5.0 weight%, 4.5 weight%, 4.0 weight%, 3.5 weight%, 3.0 weight%, 2.5 weight%, 2.0 weight%, 1.5 weight%, 1.0 weight% or 0.5 weight% water content) can be used as a nutrient composition for producing biomass according to the method of the present invention.

[0149] The biomass produced or obtained by any method in the present disclosure can be used to feed one or more organisms or provide nutrition thereto. Similar feeds are commonly used in agricultural industries in organisms such as fish, crustaceans, molluscs, poultry, pigs and cattle. Therefore, the present invention further relates to the biomass produced or obtained by any method in the present disclosure for feeding, for example, fish, crustaceans, molluscs, poultry, pigs and cattle or providing nutrition thereto. Preferably, fish include Cyprinidae, Salmonidae, Thunnini, Oreochromis and Siluriformes. Preferably, poultry include chicken (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 the present invention produces such biomass with high nutritional quality. Therefore, the present invention also relates to a biomass comprising protein obtainable by the method of the present invention, wherein the protein comprises an amino acid content comprising: a histidine content of 0.6% to 6.4% of the total biomass dry weight protein content, an isoleucine content of 1.3% to 9.2% of the total biomass dry weight protein content, a leucine content of 2.5% to 16.0% of the total biomass dry weight protein content, a lysine content of 2.0% to 14.8% of the total biomass dry weight protein content, a methionine content of 0.7% to 7.2% of the total biomass dry weight protein content, a phenylalanine content of 1.2% to 11.4% of the total biomass dry weight protein content, a threonine content of 1.1% to 9.2% of the total biomass dry weight protein content, a tryptophan content of 0.3% to 5.2% of the total biomass dry weight protein content, and a valine content of 1.1% to 12.4% of the total biomass dry weight protein content. Preferably, the amino acid content comprises a histidine content of 0.9% to 4.8% of the total biomass dry weight protein content, an isoleucine content of 2.0% to 6.9% of the total biomass dry weight protein content, a leucine content of 3.8% to 12.0% of the total biomass dry weight protein content, a lysine content of 3.0% to 11.1% of the total biomass dry weight protein content, a methionine content of 1.1% to 5.4% of the total biomass dry weight protein content, a phenylalanine content of 1.7% to 8.5% of the total biomass dry weight protein content, a threonine content of 1.6% to 6.9% of the total biomass dry weight protein content, a tryptophan content of 0.4% to 3.9% of the total biomass dry weight protein content, and a valine content of 1.7% to 9.3% of the total biomass dry weight protein content. More preferably, the amino acid content comprises a histidine content of 1.2% to 3.2% of the total biomass dry weight protein content, an isoleucine content of 2.6% to 4.6% of the total biomass dry weight protein content, a leucine content of 5.0% to 8.0% of the total biomass dry weight protein content, a lysine content of 4.0% to 7.4% of the total biomass dry weight protein content, a methionine content of 1.4% to 3.6% of the total biomass dry weight protein content, a phenylalanine content of 2.3% to 5.7% of the total biomass dry weight protein content, a threonine content of 2.1% to 4.6% of the total biomass dry weight protein content, a tryptophan content of 0.5% to 2.6% of the total biomass dry weight protein content, and a valine content of 2.2% to 6.2% of the total biomass dry weight protein content.

[0151] Furthermore, the present invention relates to a biomass obtainable by the method of the present invention, comprising a lipid content of 1.5% to 24% of the total biomass dry weight, the lipid content comprising a fatty acid content comprising a C16:0 palmitic acid content of 15% to 80% of the total biomass dry weight fatty acid content, a C16:1 palmitoleic acid content of 2.5% to 30% of the total biomass dry weight fatty acid content, and a C17:1 heptadecenoic acid content of 15% to 80% of the total biomass dry weight fatty acid content. Preferably, the lipid content is 2.3% to 18% of the total biomass dry weight, the lipid content comprising a fatty acid content comprising a C16:0 palmitic acid content of 23% to 60% of the total biomass dry weight fatty acid content, a C16:1 palmitoleic acid content of 3.8% to 22.3% of the total biomass dry weight fatty acid content, and a C17:1 heptadecenoic acid content of 23% to 60% of the total biomass dry weight fatty acid content. More preferably, the lipid content is between 3% and 12% of the total biomass dry weight, said lipid content comprising a fatty acid content comprising: a C16:0 palmitic acid content of 30% to 40% of the total biomass dry weight fatty acid content, a C16:1 palmitoleic acid content of 5% to 15% of the total biomass dry weight fatty acid content, and a C17:1 heptadecenoic acid content of 30% to 40% of the total biomass dry weight fatty acid content. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0156] Fine control of all parameters together allows for increased overall fermentation productivity by reducing and optimizing substrate limitations. Without these limitations, fermentations are capable of achieving higher productivity, and crucially, control aspects have been developed to balance this increased productivity with optimization of the protein content of the biomass.

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

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

[0159] The mixture of sample and methanol in toluene and sulfuric acid was heated under reflux for 2 hours.Fats and oils are transesterified into fatty acid methyl esters (FAMES). A small amount of normal hexane is used to extract the methyl ester mixture gained. Then anhydrous sodium sulfate is used to dry the normal hexane solution, and afterwards the sample is transferred to a chromatographic bottle. The FAMES fatty acid profile is obtained by the gas-liquid chromatography using FFAP columns (size 25m × 0.20mmID) and detected by flame ionization detector. Spectrum can be reported with or without internal standard (C17:0).

[0160] Figure 4 The range of essential amino acid amounts associated with typical soybean meal and fish meal used in animal agriculture is shown (see, for example, the US Soybean Meal Information Leaflet published by the US Soybean Export Council, accessed online on April 20, 2020: https: / / ussec.org / wp-content / uploads / 2015 / 10 / US- Soybean-Meal-Information.pdf ; and fish meal, 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), for examples of ranges of amounts of essential amino acids per 100 g of the total protein content of the isolated biomass produced according to the method of the present invention. The amino acid content of the isolated biomass produced according to the method of the present invention was determined by the following method:

[0161] The sample is oxidized in a combination of phenol, hydrogen peroxide, and formic acid. The oxidized sample is then hydrolyzed with hydrochloric acid. The amino acids are then separated using ion exchange chromatography and determined by post-column reaction with ninhydrin using photometric detection.

[0162] It can be clearly seen that the biomass produced according to the method of the present invention is of excellent quality.

[0163] Figure 5A and Figure 5BThe prediction of biomass production rate at different oxygen and hydrogen input concentration percentages is shown. The oxygen and hydrogen concentrations are the concentrations added to the liquid phase, and the biomass production rate is the production rate of biomass with at least 65% protein, given by the corresponding oxygen ( Figure 5A ) or hydrogen ( Figure 5B ) and above resulted in biomass production rates greater than 10 g / l / day. Figure 5A The amount of substrate other than oxygen is restricted so that the substrate other than oxygen is provided in a minimum amount sufficient for viability of the microorganism. Figure 5B The amount of substrate other than hydrogen is restricted so that the substrate other than hydrogen is provided in a minimum amount sufficient for viability of the microorganism.

[0164] Figure 6 Predictions of protein content by dry weight of total biomass from hydrogen-oxidizing microorganisms produced according to the present invention are shown, wherein controlling the input flow 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 Predictions of protein content by dry weight of total biomass from hydrogen-oxidizing microorganisms produced according to the present invention are shown, wherein controlling input flows includes controlling specific growth rates such that the biomass production rate is greater than 10 g / l / day.

[0166] Figure 8A Predictions of preferred hydrogen transfer rates associated with biomass production rates produced according to the present invention, where the biomass comprises at least 65% protein by dry weight relative to the total biomass, are shown. The hydrogen transfer rate required for a 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 grams of hydrogen used per gram of biomass formed. Figure 8A The necessary mass transfer required to obtain productivity exceeding 10 g / L / day, which is achievable using the method according to the invention, within the economically preferred hydrogen metabolic yield range was determined.

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

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

[0169] Example 1

[0170] In such Figure 1 The industrial chemostat bioreactor system described herein cultured according to the present invention 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] DSMZ mineral medium 81 (H-3) for chemolithotrophic growth was used, which consisted of and was prepared as follows:

[0172] Solution A:

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

[0174] Solution B:

[0175] 1.0 g NH4Cl, 0.50 g MgSO4 × 7H2O, 0.01 g CaCl2 × 2H2O, 0.005 g MnCl2 × 4H2O, 0.005 g NaVO3 × H2O, 5 ml trace element solution SL-6, 915 ml distilled water

[0176] Solution C:

[0177] Ammonium ferric citrate 0.05g, distilled water 20ml

[0178] Solutions A, B, and C were autoclaved at 121°C for 15 minutes, cooled to 50°C, and then aseptically mixed with 5.0 ml of filter-sterilized standard vitamin solution (see below). The pH of the medium was adjusted to a pH of 1 to 4 and supplemented with additional 1.5 g / L ammonium chloride, 3 × 10 -4 g / L NiCl2×6H2O and 1.5×10 -3 g / L ZnSO4×7H2O, and 1.5×10 -4 g / L CuCl2×2H2O, and 0.15 g / L ammonium ferric citrate supplementation. The above medium and preparation method can support an operating concentration of greater than 10 g / L cell dry weight and a productivity greater than 10 g / L / day of biomass (containing greater than 65% protein) under gas-limited growth conditions, wherein in situ pH control is also employed using a suitable base such as 0.2 M NaOH or NH4OH. To support higher operating cell concentrations and productivity, the medium components can be proportionally modified.

[0179] Standard vitamin solution:

[0180] Riboflavin 10mg, Thiamine-HCl×2H2O 50mg, Niacin 50mg, Pyridoxine-HCl 50mg, Calcium-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] The culture is grown for at least 3 days with constant stirring of the culture medium. Carbon dioxide gas is added to the liquid phase at a non-limiting concentration. Hydrogen and oxygen are added to the liquid phase at a ratio of 1:1 to 10:1. The gaseous substrate is optionally recirculated. The liquid culture medium is recirculated. Nutrient compounds and culture medium are added and portions of the liquid phase are removed from the system during the culture period. The total volume of the liquid phase is maintained approximately constant. The gas transfer rate is determined according to the following formula: Figure 8A and Figure 8B Indicates the preferred rate to use.

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

[0185] Table 1:

[0186]

[0187] Example 2

[0188] The culture conditions as described in Example 1 were used in Figure 1 The Copperbacterium necrotizingum strain is cultured in the industrial bioreactor system. Figure 5A The dilution rate is 2 / day, Figure 5B The dilution rate in the RT-PCR assay is 1 / day.

[0189] The result is as Figure 5A and Figure 5B The productivity shown and extrapolated from experimental results is with different hydrogen / oxygen inputs under limitation. The protein content is at least 65%.

[0190] Example 3

[0191] The culture conditions as described in Example 1 were used. Figure 1 The industrial bioreactor system was used to culture the Cupribotium necrotizer strain at a dilution rate of 1.87 / day.

[0192] The result is as Figure 6 Protein content shown and inferred from experimental results with different hydrogen:oxygen input ratios.The productivity of biomass was at a rate greater than 10 g / l / day.

[0193] Example 4

[0194] The culture conditions as described in Example 1 were used in Figure 1 The Cupribotium necrotizer strain was cultured in the industrial bioreactor system. The dilution rates at the three sites, from left to right, were 1.82 tbsp / day, 2.70 tbsp / day, and 3.53 tbsp / day, respectively. The H2:O2 ratio was 2:1.

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

[0196] Example 5

[0197] The culture conditions as described in Example 1 were used in Figure 1 The Copperbacterium necrotizingum strain is cultured in the industrial bioreactor system.

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

[0199] Example 6

[0200] The culture conditions as described in Example 1 were used in Figure 1 The Copperbacterium necrotizingum strain is cultured in the industrial bioreactor system.

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

Claims

1. A method for producing biomass comprising at least 65% protein by dry weight of the total biomass by a hydrogen-oxidizing microorganism comprising a bacterium selected from the genus Cupriavidus using one or more input streams comprising one or more gaseous substrates comprising hydrogen and oxygen, the method comprising: contacting said microorganisms in a liquid phase with a nutrient composition comprising nitrogen- and phosphorus-containing compounds and said gaseous substrate, wherein said one or more gaseous substrates comprises carbon dioxide, or wherein said nutrient composition comprises a carbon-containing compound, and wherein said input streams and nutrient compositions are controlled and wherein said biomass is produced at a rate greater than 10 g / l / day, The method further comprises: i. controlling the input flow, comprising adding hydrogen to the liquid phase in a molar ratio of 1:1 to 10:1: oxygen; ii. controlling the input flow and nutrient composition, comprising maintaining the specific growth rate of the microorganism at 1.0 / day to 8.0 / day or 0.04 / hour to 0.3 / hour; iii. controlling the input flow, comprising maintaining a hydrogen transfer rate in the liquid phase of 0.03 mol / l / hour to 1.2 mol / l / hour and / or a liquid phase oxygen transfer rate of 0.003 mol / l / hour to 0.4 mol / l / hour; iv. replacing the liquid phase of the bioreactor, wherein the volume of 4% to 30% per hour to grow and maintain the microorganisms; v. controlling the input flow comprises maintaining the liquid phase hydrogen: oxygen gas hold-up molar ratio of 0.5: 1 to 7: 1; and vi. controlling the input flow and nutrient composition includes maintaining a concentration of the microorganisms in the liquid phase of the bioreactor of at least 10 g / l. 2 . The method of claim 1 , wherein controlling the input flow and nutrient composition comprises maintaining a concentration of the microorganisms in the liquid phase of the bioreactor between 10 g / 1 and 100 g / 1.

3. The method of claim 1 , wherein controlling the input flow comprises adding a molar ratio of hydrogen to oxygen to carbon dioxide of 2 to 6: 0.85 to 2: 0.75 to 2 to the liquid phase.

4. The method of claim 1 , wherein controlling the input flow comprises maintaining a molar ratio of dissolved hydrogen:oxygen:carbon dioxide in the liquid phase of 3.183 to 12.748:0.795 to 4.25:0.75 to 2.

0.

5. The method of claim 1 , wherein controlling the input flow comprises maintaining a hydrogen concentration of 0.5 to 20 mg / l, an oxygen concentration of 0.5 to 80 mg / l, and a carbon dioxide concentration of 20 to 2000 mg / l in the liquid phase at a temperature of 28° C. to 45° C. and a vapor phase pressure of 100 to 2000 kPa.

6. The method of claim 1 , wherein controlling the input flow comprises maintaining a molar ratio of hydrogen to oxygen gas hold-up in the liquid phase of 1:1 to 3.5:

1.

7. The method of claim 1 , wherein the microorganism utilizes the hydrogen at a rate of 0.05 to 0.5 mol / g / hour, utilizes the oxygen at a rate of 0.04 to 0.18 mol / g / hour, and utilizes the carbon dioxide at a rate of 0.03 to 0.13 mol / g / hour.

8. The method of claim 1, wherein the carbon dioxide is derived from waste gas from a production or combustion process.

9. The method of claim 8, wherein the carbon dioxide is purified and concentrated to a concentration of 40% to 100% (v / v).

10. The method of claim 1, wherein contacting the microorganisms in a liquid phase with a nutrient composition comprises adding the nutrient composition, the nutrient composition being adjusted to a pH of 1 to 4 prior to addition.

11. The method of claim 10, wherein the nutrient composition comprises DSMZ medium for chemolithotrophic growth without NaHCO3.

12. The method of claim 11, wherein before addition, an additional 1.5 g / L ammonium chloride, 3 x 10 -4 g / LNiCl2×6H2O, and 1.5×10 -3 g / LZnSO4×7H2O, and 1.5×10 -4 The DSMZ medium was supplemented with 0.15 g / L CuCl2×2H2O, and 0.15 g / L ammonium ferric citrate.

13. The method of claim 1, wherein the microorganism comprises a bacterium selected from the species Cupriavidus necator.

14. Biomass obtainable by the method according to any one of claims 1 to 13, said biomass comprising protein comprising an amino acid content comprising: a histidine content of 1.2% to 3.2% of the total biomass dry weight protein content, an isoleucine content of 2.6% to 4.6% of the total biomass dry weight protein content, a leucine content of 5.0% to 8.0% of the total biomass dry weight protein content, a lysine content of 4.0% to 7.4% of the total biomass dry weight protein content, a methionine content of 1.4% to 3.6% of the total biomass dry weight protein content, a phenylalanine content of 2.3% to 5.7% of the total biomass dry weight protein content, a threonine content of 2.1% to 4.6% of the total biomass dry weight protein content, a tryptophan content of 0.5% to 2.6% of the total biomass dry weight protein content, and a valine content of 2.2% to 6.2% of the total biomass dry weight protein content.

15. Biomass obtainable by the method according to any one of claims 1 to 13, comprising a lipid content of 2.3% to 18% of the total biomass dry weight, the lipid content comprising a fatty acid content comprising: a C16:0 palmitic acid content of 23% to 60% of the total biomass dry weight fatty acid content, a C16:1 palmitoleic acid content of 3.8% to 22.3% of the total biomass dry weight fatty acid content, and a C17:1 heptadecenoic acid content of 23% to 60% of the total biomass dry weight fatty acid content.

16. Use of the biomass according to claim 14 or 15 for feeding or providing nutrition to one or more organisms.

17. The use according to claim 16, wherein the organisms include fish, crustaceans, molluscs, poultry, pigs and cattle.

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