Single-cell protein production process

Abstract

Disclosed is a process method for producing a single-cell protein, comprising the steps of: (a) providing gaseous hydrogen (H2); (b) mixing the hydrogen gas from step (a) with a carbon source to provide a C1 compound, such as methane, methanol, formaldehyde, formic acid, methanethiol, methanesulfonic acid, or a derivative thereof; (c) adding or passing the C1 compound provided in step (b) through a loop reactor comprising one or more microorganisms capable of metabolizing the C1 compound to provide an inoculated fermentation medium; (d) fermenting the inoculated fermentation medium in a fermentation process to convert the C1 compound into a biomass material; and (e) isolating the biomass material provided in step (c) to provide a second single-cell protein, wherein the carbon source is gaseous carbon monoxide (CO); gaseous carbon dioxide (CO2); or a combination thereof, or the first carbon source is an aqueous solution of carbon dioxide, such as carbonic acid, bicarbonate ion, or carbonate ion, or a combination thereof.

Description

[Technical field]

[0001] The present invention relates to an improved process for producing single cell proteins (SCPs). In particular, the present invention relates to providing an improved process for producing single cell proteins with increased simplicity, increased productivity, and / or increased efficiency, with independent location of the fermentation process (and fermentation reactor) from natural gas recovery, independent from fossil fuel cost fluctuations, reduced environmental and / or atmospheric impact. [Background technology]

[0002] Due to the increase in the world population, people have an increasing demand for protein-rich diets, and animals (pets or livestock) and fish are being fed more and more protein-rich feed to promote faster growth and development, and for the health of the animals.

[0003] However, with a growing world population and increasing demand for protein in the livestock and pet industries, there is strong evidence that agriculture will not be able to meet this demand, posing a serious risk of food shortages.

[0004] Industrial agriculture is notable for its high water footprint, high land use, destruction of biodiversity and general environmental degradation, and contributes to climate change by producing around one-third of all greenhouse gas emissions.

[0005] To meet the increasing protein demand and at least some of the stated shortcomings of industrial agriculture, the production of single-cell proteins (SCPs) has been shown to be a very interesting candidate.

[0006] Single cell proteins (SCPs) can be grown by fermentation of biomass through the growth of microorganisms on hydrocarbon, nitrogen, and other substrates. SCP production represents a fail-safe option for mass food production that can reliably produce food worldwide, even in harsh climatic conditions.

[0007] The SCP product can be used directly in a food or feed product, for example as a liquid product or as a spray-dried product. Alternatively, the SCP or its biomass can be further processed, for example by hydrolysis and / or separation, to provide special fractions, remove impurities, or concentrate components prior to use in a food or feed product.

[0008] The microorganisms traditionally used for the production of SCP are methylotrophic or methanotrophic microorganisms, which digest methane provided in the form of natural gas (as carbon source gas) in the presence of oxygen and nitrogen compounds and convert it into biomass as the SCP product.

[0009] Methane is the main component of natural gas, accounting for about 87% by volume. Its primary source is extraction from geological formations. It is associated with other hydrocarbon fuels. Generally, natural gas-producing deposits are buried deeper and at higher temperatures than oil-bearing deposits, which can make recovery more difficult. Methane is generally transported in bulk by pipeline in the form of natural gas, or in liquefied form by LNG carriers, or in a few countries by truck.

[0010] Thus, challenges with currently offered SCP delivery processes include that the process location may be limited to areas where methane or natural gas is available, or that methane or natural gas needs to be transported to the SCP fermenter, which adds to the production costs, and, as mentioned above, the methane used is traditionally obtained from fossil fuels, which can be a limiting factor, subject to large fluctuations in cost and harmful effects on the environment and atmosphere.

[0011] Furthermore, fermentation processes based on the digestion of natural gas involve the co-fermentation of different types of microorganisms, and natural gas contains small amounts of different hydrocarbons other than methane that need to be digested so as not to accumulate in the fermentation medium, reducing the efficiency of the fermentation process or even stopping and then resuming the fermentation process.

[0012] Thus, there is a need in the industry for improved processes for producing single-cell proteins, and an improved process that overcomes the problems of the prior art would be advantageous.

[0013] In particular, there is a need for improved processes that are independent of the location of natural gas extraction, i.e., the availability of cheap methane gas that is not affected by fluctuations in fossil fuel prices, and that are more environmentally and / or air-friendly, simpler to process, more efficient, and / or more reliable would be advantageous. Summary of the Invention

[0014] Accordingly, an object of the present invention relates to an improved process for producing single-cell proteins, and an improved process which overcomes the problems of the prior art would be advantageous.

[0015] In particular, it is an object of the present invention to provide an improved process for producing single-cell proteins that solves the above-mentioned problems of the prior art regarding the location of the fermentation process (and fermentation reactor), the variability in the cost of fossil fuels, environmental and / or atmospheric challenges, simplicity, productivity, and efficiency.

[0016] Thus, one aspect of the present invention relates to a process for providing a first reaction product by a first fermentation process carried out in a first loop reactor, the method comprising the steps of: (i) adding an inoculum comprising one or more methanogenic microorganisms to a first loop reactor to provide a first inoculum fermentation medium; (ii) adding gaseous hydrogen (H2) to the first inoculation fermentation medium; (iii) adding a first gaseous carbon source, such as gaseous carbon monoxide (CO); gaseous carbon dioxide (CO2) or a combination thereof, to the first inoculated fermentation medium, or adding a non-gaseous carbon source, such as an aqueous solution of carbon dioxide, such as carbonic acid, bicarbonate ions or carbonate ions, or a combination thereof; (iv) fermenting the first fermentation medium to provide a first reaction product; and (v) isolating the first reaction product provided in step (iv).

[0017] Another second aspect of the present invention relates to a process for producing a single-cell protein comprising the steps of: (a) providing gaseous hydrogen (H2); (b) mixing the hydrogen gas from step (a) with a gaseous carbon source (gaseous carbon monoxide (CO); gaseous carbon dioxide (CO2); or a combination thereof, or the first carbon source is an aqueous solution of carbon dioxide, such as carbonic acid, bicarbonate ions, or carbonate ions, or a combination thereof) to provide a C1 compound; (c) adding or passing the C1 compound provided in step (b) through a loop reactor containing one or more microorganisms capable of metabolizing said C1 compound to provide an inoculated fermentation medium; (d) fermenting the second inoculated fermentation medium in a fermentation process to convert said C1 compounds into biomass material; and (e) isolating the biomass material provided in step (c) to provide single cell proteins.

[0018] Yet another third aspect of the present invention relates to a loop reactor comprising a loop section and a top tank, said loop section comprising a downflow section connected to an upflow section via a U-shaped section, said loop section comprising at least one inlet for injecting gaseous hydrogen (H2).

[0019] A further fourth aspect of the invention relates to a single-cell protein composition comprising a first single-cell protein, as described in more detail below, and a second single-cell protein, as described in more detail below.

[0020] Furthermore, a fifth aspect of the present invention relates to the use of the single-cell protein composition according to the present invention as an ingredient in a feed product for animals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present invention will now be described in more detail.

[0022] The inventors of the present invention have found that currently available processes for providing single cell proteins (SCPs) have several undesirable limitations, undesirable drawbacks, and challenges that negatively affect the use of the technology and the productivity of the process for producing single cell proteins (SCPs). Therefore, the inventors of the present invention have surprisingly discovered a process for decoupling the process from a site having an available carbon source (e.g., methane), which also represents a more environmentally and / or atmospherically friendly process, making the process more convenient and / or more efficient.

[0023] First aspect of the present invention A preferred embodiment of the present invention relates to a process for providing a first reaction product by a first fermentation process carried out in a first loop reactor, the method comprising the following steps: (i) adding an inoculum comprising one or more methanogenic microorganisms to a first loop reactor to provide a first inoculum fermentation medium; (ii) adding gaseous hydrogen (H2) to the first inoculation fermentation medium; (iii) adding a first gaseous carbon source, such as gaseous carbon monoxide (CO), gaseous carbon dioxide (CO), or a combination thereof, to the first inoculum fermentation medium; (iv) fermenting the first fermentation medium to provide a first reaction product; and (v) isolating the first reaction product provided in step (iv).

[0024] In the context of the present invention, the term "loop" refers to a loop reactor comprising a loop section and a top tank (gas-liquid separation tank). The top tank may include a vent tube for discharging exhaust gas from the top tank. The loop section is composed of a substantially vertical downflow section connected to a substantially vertical upflow section via a horizontal or U-shaped section.

[0025] In a preferred embodiment of the invention, the loop, if present in the fermenter, comprises a circulation pump for circulating the fermentation medium.

[0026] In a further embodiment of the invention, the loop portion has a length which may be longer, preferably substantially longer, than the length and / or height of the top tank.

[0027] In a further embodiment of the invention, the top tank comprises a volume larger than the volume of the loop. Preferably, the fermentation reactor comprises a loop having a length longer, preferably substantially longer, than the length and / or height of the top tank, and the top tank comprises a volume larger than the volume of the loop.

[0028] In one embodiment of the present invention, the loop portion of the present invention may be associated with at least one downflow portion, at least one upflow portion, and at least one connection portion.

[0029] In this specification the term "U-part" relates to a bend at the bottom of a fermentation reactor or loop reactor, connecting the lower ends of the upflow and downflow sections.

[0030] Preferably, the one or more upflow sections and the one or more downflow sections are vertical or substantially vertical.

[0031] A loop reactor according to the invention can be designed as a vertical loop reactor, or a horizontal loop reactor, or a "tilted" loop reactor. In general, a horizontal loop reactor is characterized by having essentially the same hydrostatic pressure in all parts of the loop, whereas a vertical or tilted loop reactor has a pressure profile in which the hydrostatic pressure varies in different parts of the reactor.

[0032] In one embodiment of the invention, the fermentation reactor may be a vertical loop reactor. A vertical loop reactor may refer to a loop reactor having a main part of a U-shaped section in a vertical or substantially vertical position relative to a horizontal position. In one embodiment of the invention, the fermentation reactor comprises a main part of a U-shaped section that is vertically or substantially vertically arranged.

[0033] In another embodiment of the invention, the fermentation reactor may be a horizontal loop reactor. A horizontal loop reactor may refer to a loop reactor in which the main part of the U-shaped section is in a horizontal or substantially horizontal position as opposed to a vertical position. In one embodiment of the invention, the fermentation reactor comprises a main part of the U-shaped section in a horizontal or substantially horizontal position.

[0034] Preferably, the fermentation reactor can be designed as a vertical or at least inclined loop reactor.

[0035] In the context of the present invention, the term "major portion" relates to at least 51% (v / v); at least 55% (v / v), etc.; for example at least 60% (v / v); at least 65% (v / v), etc.; for example at least 70% (v / v); at least 75% (v / v); for example at least 80% (v / v); at least 85% (v / v), etc.; for example at least 90% (v / v); at least 95% (v / v), etc.; for example at least 98% (v / v).

[0036] As used herein, the term "top tank" refers to a vessel located at the top of a fermentation reactor and responsible for removing exhaust gases from the fermentation liquor. Preferably, the top tank is only partially filled with fermentation liquor during operation / fermentation. In one embodiment of the present invention, the term "partially filled with fermentation liquor" refers to a ratio of fermentation liquor to gases of 90:10; 80:20, etc.; such as 70:30; 60:40, etc.; such as 50:50; 40:60, etc.; such as 30:70; 20:80, etc.; such as 10:90.

[0037] In a preferred embodiment, exhaust gas removal involves capturing CO and / or CO2 and injecting it into the part of the loop reactor where reaction with H2 takes place. See below for details.

[0038] In the context of the present invention, "visual inspection means" relates to one or more means that allow a person skilled in the art to obtain direct information regarding, for example, the flowability and / or foaming properties in the top tank and / or in the loop part.

[0039] In one embodiment of the present invention, the direct information can be real-time information regarding the foaming characteristics in the top tank.

[0040] In a further embodiment of the present invention, the first carbon source may be a first gaseous carbon source; or a first liquid carbon source. Preferably, the first carbon source is a first gaseous carbon source.

[0041] The first gaseous carbon source may be gaseous carbon monoxide (CO); gaseous carbon dioxide (CO2); or a combination thereof. Alternatively, the first carbon source may be an aqueous solution of carbon dioxide, such as carbonic acid, bicarbonate ions, carbonate ions, or a combination thereof. In other words, in these cases, the carbon dioxide is dissolved in water and is in equilibrium with the carbonic acid and its deprotonated ions.

[0042] Gaseous Hydrogen (H2) and / or A first gaseous carbon source, such as gaseous carbon monoxide (CO); gaseous carbon dioxide (CO2); or a combination thereof, or an aqueous solution of carbon dioxide, such as carbonic acid, bicarbonate ions, or carbonate ions, or a combination thereof. The addition or inflow to the inoculum fermentation medium present in the first loop reactor can be controlled by the need for hydrogen (H2) required for optimized production and / or the hydrogen (H2) consumption of one or more methanogenic microorganisms. As described below, the first carbon source in the form of (optionally dissolved) CO and / or CO2 can be provided from a process in which C1 compounds are metabolized into biomass by methanotrophic or methylotrophic microorganisms.

[0043] The cultivation and fermentation of methanogenic microorganisms is generally known to those skilled in the art.

[0044] In one embodiment of the invention, a first gaseous carbon source, such as gaseous hydrogen (H2) and / or gaseous carbon monoxide (CO); gaseous carbon dioxide (CO2); or a combination thereof, or an aqueous solution of carbon dioxide, such as carbonate or bicarbonate ions or carbonate ions, or a combination thereof, can be added continuously to the first inoculum fermentation medium during the fermentation process.

[0045] In the context of the present invention, the term "hydrogen" relates to the chemical compound dihydrogen (H2). Hydrogen (H2) can be provided in gaseous form.

[0046] In one embodiment of the present invention, gaseous hydrogen may be provided from the electrolysis of water; obtained from natural sources, such as terrestrial sources; produced by microorganisms; or produced chemically.

[0047] Water electrolysis involves the passing of an electric current which splits water molecules into oxygen and hydrogen gas. A direct current power source connected to two electrodes, or plates (usually made from an inert metal such as platinum or iridium), is placed in water and the hydrogen gas can be easily collected from the cathode.

[0048] Preferably, natural water, such as seawater, is electrolyzed using electrical current generated from sustainable energy sources such as wind, wave, tidal, solar, geothermal, hydroelectric, some of which are "unstable" in the sense that the energy output depends on conditions (e.g. wind speed and wave height at sea), and utilising excess energy from such sources in times of overproduction is a commonly known problem. Storing energy in the form of hydrogen is a known solution to this storage problem, and the present invention provides a way to utilise such stored hydrogen.

[0049] In one embodiment of the invention, the first gaseous carbon source, such as gaseous carbon monoxide (CO) and / or gaseous carbon dioxide (CO2) and / or an aqueous solution of carbon dioxide, such as carbonate or bicarbonate ions or carbonate ions, or a combination thereof, can be obtained from a carbon capture process, or a chemical, enzymatic or microbial process.

[0050] The methanogenic microorganism can be a methanogenic archaea, a methanogenic bacterium, a methanogenic yeast, a methanogenic fungus, or a combination thereof.

[0051] In one embodiment of the present invention, the methanogenic microorganism may be a prokaryote. Preferably, the methanogenic microorganism may be a methanogenic archaea.

[0052] The methanogenic archaea is preferably selected from the group consisting of Methanobacterium bryantii, Methanobacterium formicum, Methanobacterium thermoalcaliphium, Methanothermobacter wolfeii, Methanobrevibacter smithii, Methanobrevibacter ruminantium, Methanococcus voltae, Methanomicrobium mobile, Methanolacinia paynteri, Methanospirillum hungatei, Methanosarcina acetivorans ... acetivorans; Methanosarcina barkeri; Methanosarcina mazei; Methanosarcina thermophile; Methanococcoides methylutens; Methanosaeta concilii(soehngenii); and Methanosaeta thermophila.

[0053] In one embodiment of the invention, the fermentation process may be batch fermentation, fed-batch fermentation, or continuous fermentation. Preferably, the fermentation process may be continuous.

[0054] For example, for commercial production of SCP, the fermentation process may include the following three fermentation stages: Batch fermentation; in which the organisms are grown first, all materials except the desired organisms are decontaminated in an autoclave, and then charged into the reactor with the organisms to start the process. The organisms used go through all the growth phases (lag phase, logarithmic or exponential phase, stationary phase). In this mode of operation, conditions change continuously with time in a non-steady-state system, requiring a lot of effort and involvement. Fed-batch fermentation; a biotechnological operating process in which one or more nutrients are provided to a bioreactor during cultivation and the product remains in the bioreactor until termination. Fed-batch fermentation can traditionally be performed after batch fermentation to achieve very high cell concentrations of the organism before moving to continuous fermentation, which requires inhibitory high concentrations of nutrients and is very difficult or even impossible. Fed-batch fermentation can be used to prepare cell cultures for continuous fermentation. Continuous fermentation: a production model of a fermentation process in which the microorganisms are fed with the sterile fermentation medium used to grow them, while at the same time removing part of the fermentation medium containing the biomass from the system, thus offering the unique feature of being able to continuously feed biomass either as single cell proteins or fractionated into various fractions.

[0055] The production mode of the process according to the invention can preferably be carried out as a continuous fermentation process. Preferably, the continuous fermentation process can be started by adding water, necessary nutrient salts and microorganisms to the fermentation reactor to prepare a first inoculum fermentation medium, followed by a batch fermentation process and / or a fed-batch fermentation process, and then the batch fermentation process and / or the fed-batch fermentation process can be started. When a sufficient amount of biomass is reached, the continuous fermentation process can be started.

[0056] For economic reasons, there may be an interest in starting continuous stationary fermentation as soon as possible to save time and costs and get SCP products to market sooner and more profitably, and also to be able to determine the relationship between environmental conditions and microbial behavior, including genetic and phenotypic expression.

[0057] The first inoculum fermentation medium can be fermented during batch and / or fed-batch fermentation for a period ranging from 6 hours to 6 days; such as for a period of 12 hours to 5 days; for example, for a period of 1 to 4 days, for a period of 2 to 3 days, etc.

[0058] The first inoculum fermentation medium can be circulated into the first fermentation reactor, preferably by a first pressure control device, and the addition of substrates such as gaseous hydrogen (H2) and a carbon source can be started to initiate the first fermentation process. When the density of the microorganisms reaches a concentration of about 0.5-10%, preferably 1-5% (dry weight), the first fermentation process can be transitioned to a continuous fermentation process in which the first inoculum fermentation medium is continuously withdrawn from the first fermentation reactor, for example from a top tank and / or from the U-shaped section, for downstream processing to provide the desired first reaction product. At the same time, the first inoculum fermentation medium can be continuously withdrawn from the first fermentation reactor and substrates including water, salts, and nutrients can be added.

[0059] In one embodiment of the invention, during continuous fermentation, the first inoculum fermentation medium is allowed to ferment for a period of at least 3 days, such as at least 6 days, such as at least 2 weeks, such as at least 4 weeks, such as at least one and a half months, such as at least 2 months, such as at least 3 months.

[0060] The first inoculum fermentation medium may ferment during continuous fermentation until the culture is forcibly or manually stopped due to maintenance needs; microbial contamination; chemical contamination; substrate problems, and the like.

[0061] In one embodiment of the invention, the first inoculum fermentation medium may be fermented at a temperature in the range of 25-60°C; such as in the range of 30-50°C; for example in the range of 35-45°C; such as in the range of 40-43°C.

[0062] The first fermentation process involves the fermentation of a methanogenic microorganism to provide a first reaction product.

[0063] In the context of the present invention, the term "first reaction product" relates to one or more products resulting from the first fermentation process by the action of methanogenic microorganisms.

[0064] In one embodiment of the invention, the first reaction product provided in step (v) may be a first biomass matter; a first single-cell protein; a C1 compound; or a combination thereof.

[0065] In a further embodiment of the invention, the first reaction product comprises a single-cell protein.

[0066] The first biomass material and / or the first single-cell protein may include one or more methanogenic microorganisms.

[0067] In one embodiment of the present invention, the C1 compound may be methane, methanol, formaldehyde, formic acid, methanethiol, methanesulfonic acid, or derivatives thereof. Generally, when C1 compounds are mentioned throughout this disclosure, these are preferred embodiments thereof, and preferably, the C1 compound is methane.

[0068] Preferably, a plurality of first reaction products may be obtained from the first fermentation process.

[0069] In one embodiment of the invention, the first reaction product provided in step (v) may comprise a combination of the first single-cell protein and the C1 compound.

[0070] The first reaction product may comprise a C1 compound, and the C1 compound (defined above) may be added to a second loop reactor, the second loop reactor comprising a second inoculated fermentation medium, the second inoculated fermentation medium comprising one or more microorganisms capable of metabolizing the C1 compound and converting the C1 compound to a second reaction product by a second fermentation process.

[0071] Alternatively, the C1 compounds are reacted in a first loop reactor that also contains a second inoculated fermentation medium, which contains one or more microorganisms capable of metabolizing the C1 compounds and converting them to a second reaction product by a second fermentation process. This may be carried out in a compartment or zone of the first loop reactor different from the compartment or zone in which the process producing the C1 compounds is carried out (this is particularly relevant when the process is continuous). It may also be carried out in another subsequent time cycle after the process producing the C1 compounds is carried out. In this case, the entire loop reactor needs to be recalibrated for the conversion of the C1 compounds.

[0072] In the context of the present invention, the term "second reaction product" relates to one or more products resulting from the second fermentation process by the action of one or more microorganisms capable of metabolizing C1 compounds.

[0073] The second reaction product may be a second single cell protein, a second biomass matter, CO2, or a combination thereof.

[0074] The second reaction product may be a second single cell protein, a second biomass material, or a fraction thereof.

[0075] In one embodiment of the invention, the second reaction product may be a combination of CO2, single cell protein, or a fraction of a single cell protein.

[0076] The single cell protein fraction or biomass product fraction may be obtained by the methods described in WO 2018 / 115042, as well as downstream processing of the first and / or second reaction products, which may be carried out according to the processes described in WO 2018 / 115042.

[0077] The one or more microorganisms capable of metabolizing C1 compounds may be one or more aerobic microorganisms.

[0078] In one embodiment of the invention, the one or more aerobic microorganisms may be one or more aerobic methanotrophic microorganisms and / or one or more aerobic methylotrophic microorganisms. Preferably, the one or more aerobic methanotrophic microorganisms or the one or more aerobic methylotrophic microorganisms may be one or more aerobic methanotrophic bacteria and / or one or more aerobic methylotrophic bacteria, respectively.

[0079] In further embodiments of the invention, the one or more microorganisms capable of metabolizing a C1 compound may not be a recombinant microorganism.

[0080] In the context of the present invention, the term "recombinant microorganism" relates to a genetically modified organism (GMO) whose genetic material has been modified using genetic engineering techniques. Recombinant microorganisms may be considered in contrast to genetic modifications that occur naturally in microorganisms, for example by mating and / or natural recombination.

[0081] Preferably, the one or more microorganisms that metabolize C1 compounds may be one or more naturally occurring microorganisms.

[0082] In one embodiment of the invention, the one or more microorganisms capable of metabolizing C1 compounds can be bacteria, such as methanotrophic bacteria or methylotrophic bacteria; yeast, such as methanotrophic yeast or methylotrophic yeast; fungi, such as methanotrophic fungi or methylotrophic fungi; or combinations thereof.

[0083] In the context of the present invention, the term "naturally occurring microorganism" relates to a microorganism whose genetic material has not been modified using genetic engineering techniques. Natural modifications and changes in the genetic material of a microorganism may be covered by the term "naturally occurring microorganism".

[0084] In one embodiment of the present invention, the one or more aerobic methanogenic bacteria may be Methylococcus. Preferably, the Methylococcus is M. capsulatus, more preferably, the M. capsulatus is M. capsulatus (Bath), and even more preferably, the M. capsulatus (Bath) identified in NCIMB 11132.

[0085] In a further embodiment of the invention, one or more microorganisms capable of metabolizing C1 compounds may be provided in combination with another microorganism (as in co-fermentation).

[0086] The other microorganisms in the co-fermentation may be selected depending on impurities, such as carbon compounds other than C1, that are not metabolized or digested by the one or more microorganisms capable of metabolizing C1 according to the present invention and thus may accumulate in the second inoculum fermentation medium during the second fermentation process.

[0087] In one embodiment of the present invention, the co-fermentation may be provided by combining one or more microorganisms capable of metabolizing C1, preferably M. capsulatus with one or more microorganisms selected from Ralstonia sp.; Bacillus brevis; Brevibacillus agri; Alcaligenes acidovorans; Aneurinibacillus danicus, and Bacillus firmus.

[0088] In particular, the co-fermentation according to the invention may relate to a co-fermentation comprising a combination of M. capsulatus (preferably, NCIMB 11132); A. acidovorans (preferably, NCIMB 13287); B. firmus (preferably, NCIMB 13289); and A. danicus (preferably, NCIMB 13288).

[0089] In one embodiment of the present invention, the yeast may be a methanotrophic yeast or a methylotrophic yeast. Preferably, the yeast may be selected from Pichia pastoris, Komagataella phaffii, Komagataella pastoris, and / or Komagataella pseudopastoris.

[0090] In one embodiment of the invention, the second biomass material and / or the second single-cell protein may comprise one or more methanotrophic microorganisms and / or one or more methylotrophic microorganisms.

[0091] In a further embodiment of the invention, the first single-cell protein and the second single-cell protein may be mixed to provide a composite single-cell protein.

[0092] In one embodiment of the invention, the second inoculum fermentation medium can be fermented during batch fermentation for a period ranging from 6 hours to 6 days; such as for a period of 12 hours to 5 days; for example, for a period of 1 to 4 days, such as for a period of 2 to 3 days.

[0093] During production mode, the second fermentation process according to the invention can be preferably carried out as a continuous fermentation process. Preferably, the continuous fermentation process of the second inoculum fermentation medium can be started by adding water, necessary nutrient salts, and microorganisms (including one or more microorganisms capable of metabolizing C1) to a second fermentation reactor producing the second inoculum fermentation medium following a batch and / or fed-batch fermentation process, and the batch and / or fed-batch fermentation process can be started.

[0094] The second inoculum fermentation medium is preferably circulated into the fermentation reactor by a first pressure control device, and the addition of substrates, such as gaseous C1 compounds, can be initiated to initiate fermentation. When the density of microorganisms in the second fermentation reactor reaches a concentration of about 0.5-10%, preferably 1-5% (dry weight), the second fermentation process can be transitioned to a continuous fermentation process in which the second inoculum fermentation medium is continuously withdrawn from the second fermentation reactor, for example from a top tank and / or U-shaped section, and subjected to downstream processing to provide the desired second reaction product. At the same time, substrates, including water, salts and nutrients, can be added while the second inoculum fermentation medium is continuously withdrawn from the fermentation reactor.

[0095] In one embodiment of the invention, the second inoculum fermentation medium is allowed to ferment for a period of at least 3 days, such as at least 6 days, such as at least 2 weeks, such as at least 4 weeks, such as at least one and a half months, such as at least 2 months, such as at least 3 months, during the continuous fermentation.

[0096] The second inoculum fermentation medium is allowed to ferment during the continuous fermentation until the culture is forcibly or manually stopped due to maintenance needs, microbial contamination, chemical contamination, substrate problems, or the like.

[0097] In one embodiment of the invention, the second inoculum fermentation medium may be fermented at a temperature in the range of 25-60°C; such as in the range of 30-50°C; for example in the range of 35-45°C; such as in the range of 40-43°C.

[0098] In a further embodiment of the invention, the second fermentation process may include the addition of carbon dioxide (CO2) to the second inoculated fermentation medium.

[0099] In one embodiment of the present invention, one or more methanotrophic microorganisms and / or one or more methylotrophic microorganisms according to the present invention may be added to a first inoculated fermentation medium to provide a co-fermentation between one or more methanogenic microorganisms; and one or more methanotrophic microorganisms and / or one or more methylotrophic microorganisms. The one or more methanotrophic microorganisms and / or one or more methylotrophic microorganisms may then convert the C1 compounds produced from the first fermentation process directly from the first inoculated fermentation medium prior to the isolation step (v).

[0100] In a further embodiment of the present invention, gaseous oxygen (O2) can be added to the second inoculated fermentation medium.

[0101] As previously described with respect to the first fermentation process, hydrogen (H2) is added to the first fermentation reactor, where the hydrogen (H2) may be provided from the electrolysis of water, which is broken down into oxygen (O2) gas and hydrogen (H2) gas by the passage of an electric current.

[0102] In one embodiment of the invention, gaseous oxygen (O2) is provided from the hydrolysis of water to produce gaseous hydrogen (H2), which can be added to a first inoculated fermentation medium and gaseous oxygen (O2) can be added to a second inoculated fermentation medium.

[0103] When gaseous hydrogen is provided from the electrolysis of water, oxygen is also obtained, which can be used in a second fermentation process to provide a second reaction product, for example a second single-cell protein containing methanotrophic or methylotrophic microorganisms.

[0104] In one embodiment of the invention, the CO produced in the second fermentation process can be recycled to the first inoculation fermentation medium and / or the second inoculation fermentation medium.

[0105] Second Aspect of the Invention A preferred embodiment of the present invention relates to a process for producing a second single-cell protein comprising the steps of: (a) providing gaseous hydrogen (H2); (b) mixing the hydrogen gas from step (a) with a first carbon source, as discussed in detail above in relation to the first aspect of the invention and embodiments thereof, to provide a C1 compound, as discussed in detail above in relation to the first aspect of the invention and embodiments thereof; (c) adding or passing the C1 compound provided in step (b) through a loop reactor containing one or more microorganisms capable of metabolizing the C1 compound to provide an inoculated fermentation medium; (d) fermenting the second inoculated fermentation medium in a fermentation process to convert the C1 compounds into a second biomass material; and (e) isolating the biomass material provided in step (c) to provide a second single-cell protein.

[0106] In one embodiment of the present invention, the C1 compound provided in step (b) is not only obtained according to the first fermentation process described above, but also by any cell-free reaction between H2 and a first carbon source, or by any process involving fermentation of living cells, such as methanogenic bacteria. Thus, in these embodiments, the exact method of production of the C1 compound is not limited to fermentation-based production, or even to production in a loop reactor.

[0107] An example of a cell-free reaction would be the Sabatier reaction CO2 + 4H2 → CH4 + 2H2O, which was originally performed using a nickel catalyst. Ruthenium, alumina and nickel are well-known examples, but other catalysts are available to those skilled in the art. Enzymes are also known to catalyze useful reactions, formate dehydrogenase and carbon dioxide reductase are both enzymes that facilitate the production of formic acid from CO2. A useful review of available reactions can be found in Porosoff MD et al. 2016, Energy & Environmental Science, Issue 1: doi.org / 10.1039 / C5EE02657A.

[0108] A useful review on the potential for enzyme-catalyzed reactions between H2CO2 and CO is provided by Shi J et al. 2015, Chemical Society Reviews, issue 17: "Enzymatic conversion of carbon dioxide", doi.org / 10.1039 / C5CS00182J.

[0109] One convenient way to improve the overall efficiency of the presently disclosed process is to feed the CO and / or CO produced in the process back into the above-described process of reacting the carbon source with H.

[0110] For economical considerations, all steps (a)-(e) can also be carried out in the same loop reactor, which can be achieved by having the loop reactor comprise different compartments or zones for carrying out steps b and d; or the loop reactor is operated in time-separated cycles, including at least one cycle carrying out step b and at least one cycle carrying out step d.

[0111] In a further embodiment of the present invention, the gaseous hydrogen gas (H2) provided in step (a) can be obtained by subjecting water to a water splitting process, which causes water molecules (H2O) to split into a hydrogen gas (H2) fraction and an oxygen gas (O2) fraction.

[0112] Preferably, the water decomposition process is electrolysis.

[0113] Electrolysis is the process of connecting a power source to two electrodes or plates (usually made of an inert metal such as platinum or iridium) placed in water. When the power source is activated, hydrogen (H2) appears at the cathode (where the electrons enter the water) and oxygen appears at the anode. Assuming ideal Faradaic efficiency, the amount of hydrogen evolved is twice the amount of oxygen, and both are proportional to the total charge conducted by the solution.

[0114] During the electrolysis of water, oxygen appears at the anode and can be isolated and added to the second fermentation medium.

[0115] Carbon dioxide (CO2) can be produced from the second fermentation process, as described herein, and can be recycled.

[0116] Third aspect of the present invention A preferred embodiment of the present invention relates to a loop reactor comprising a loop section and a top tank, said loop section comprising a downflow section connected to an upflow section via a horizontal section, a substantially horizontal section or a U-shaped section, the loop section comprising at least one inlet for injecting gaseous hydrogen (H2).

[0117] In one embodiment of the invention, the loop section may further comprise at least one inlet for injecting gaseous carbon monoxide (CO); gaseous carbon dioxide (CO2), or a combination thereof, or alternatively an aqueous solution of carbon dioxide, such as carbonic acid, bicarbonate ions, carbonate ions, or a combination thereof. In particular, the loop reactor further comprises a system for injecting CO2 and / or CO to react with H2, the loop reactor preferably comprising a compartment or zone for the reaction of H2 with CO2 and / or CO, said compartment optionally comprising at least one catalyst, such as an enzyme, for catalysing the reaction, see above for details regarding enzymes and catalysts. The catalyst or enzyme can be made available for the reaction in several ways. Typically, a surface is coated with what is constituted by the catalyst or enzyme, and such surfaces can take any convenient form, such as the surface of a fibre or tube (both internally and externally, the surface of a bead, etc.).

[0118] Preferably, the loop reactor comprises a circulation pump.

[0119] In one embodiment of the present invention, a first pressure control device can be provided in the loop portion of the loop reactor. Preferably, a circulation pump can function as the first pressure control device.

[0120] The first pressure control device may be located at the top of the downflow section of the loop section of the loop reactor.

[0121] A second pressure regulator may be provided downstream of the first pressure regulator, and is preferably provided at an upper portion of the upflow section.

[0122] The second pressure control device may be selected from the group consisting of a narrowing of the diameter / cross-section of the upper part of the upflow section; a plate with holes; a jet; a nozzle; a valve; a hydrocyclone; or a pump (such as a propeller pump, a lobe pump, a turbine pump, etc.).

[0123] The first pressure controller can pump the fermentation medium towards the second pressure controller, which creates an increased pressure in the fermentation medium between the first and second pressure controllers. This increase in pressure can increase the mass transfer of gas from an undissolved state to a dissolved state, making it available for consumption by the microorganisms.

[0124] In one embodiment of the present invention, the loop reactor may include at least one inert mixer and / or at least one active mixer.

[0125] The top tank of the loop reactor may contain: (i) a first outlet connecting the top tank and the downflow section of the loop and allowing the fermentation broth present in the top tank to flow from the top tank into the loop; (ii) a first inlet connecting the top tank and the upflow section of the loop, allowing the fermentation liquid present in the loop to flow from the loop into the top tank; and

[0126] The top tank may further include a vent tube for venting exhaust gases from the top tank.

[0127] The top tank, or other relevant part of the loop reactor, may further include a system for capturing the CO and / or CO2 produced in the reactor, which is coupled to a system for injecting the CO and / or CO2 produced in the reactor to react with H2, thus providing a practical implementation of the CO2 recycling discussed above.

[0128] In one embodiment of the present invention, the top tank further includes a visual inspection means.

[0129] In a further embodiment of the invention, the loop portion includes a visual inspection means.

[0130] A visual inspection means can be provided in the loop section to control the flow of fermentation medium and / or turbulence of the fermentation medium in the loop section to ensure optimized fermentation and increased productivity of the fermentation process.

[0131] A visual inspection means may be provided in the top tank to control foaming and / or turbulence of the fermentation broth in the top tank and ensure optimal degassing of the exhaust gases and thus increased productivity of the fermentation process.

[0132] Preferably, the visual inspection means is positionable within the top tank with a horizontal or substantially horizontal inspection field.

[0133] The visual inspection means can be disposed on the side of the top tank so as to be able to see both above and below the fermentation liquid surface.

[0134] Preferably, the visual inspection means can be located at the end of the top tank.

[0135] Preferably, the visual inspection means may be located at an end of the top tank providing a field of view from the first inlet (or upflow section) towards the first outlet (or downflow section).

[0136] In one embodiment of the present invention, the visual inspection means according to the present invention may be an inspection hole, a camera, or a combination of an inspection hole and a camera, such as an in-line camera.

[0137] In a further embodiment of the present invention, the inspection hole may be a sight glass.

[0138] The loop reactor may include at least one hydrogen (H2) sensor, which may provide information about the amount of dissolved and / or undissolved hydrogen (H2) in the first inoculated fermentation medium, thus allowing the optimization of the first fermentation process according to the invention.

[0139] Further details of suitable modifications to the loop reactor and features as to how to operate such a loop reactor, as well as the processing of the resulting biomass, may be as described in WO 2010 / 069313; WO2000 / 70014; WO2003 / 016460; WO2018 / 158319; WO2018 / 158322; WO2018 / 115042 and WO2017 / 080987, all of which are incorporated by reference.

[0140] Fourth aspect of the present invention A preferred embodiment of the present invention relates to a composite single-cell protein composition comprising a first single-cell protein according to the present invention and a second single-cell protein according to the present invention.

[0141] Preferably, the first single-cell protein comprises one or more methanogenic microorganisms.

[0142] Preferably, the second single-cell protein comprises one or more methanotrophic or methylotrophic microorganisms.

[0143] In one embodiment of the invention, the complex single-cell protein comprises a combination of: One or more methanogenic microorganisms; and One or more methanotrophs or one or more methylotrophs.

[0144] Fifth aspect of the present invention A preferred embodiment of the present invention relates to the use of the complex single-cell protein composition according to the invention as an ingredient in an animal feed product or a human food product.

[0145] The feed product may be a ruminant feed product, a fish feed product, a swine feed product or a poultry feed product.

[0146] In this disclosure, the focus is on loop reactor technology. However, it will be understood that embodiments of the invention that integrate the production of C1 compounds (by any convenient method) and the subsequent production of biomass (such as single-cell proteins) by fermentation do not necessarily have to be carried out in a loop reactor. The type of fermentation reactor is not important, but a loop reactor is preferred. Also, systems that include a combination of a reaction chamber and a fermentation reactor can be used, especially when the reaction of H2 and CO2 is carried out in a cell-independent / cell-free system. Thus, in a broad embodiment of the invention, the reactor for carrying out the fermentation is a fermentation tank of any possible configuration, but the reaction of H2 and CO2 can be carried out in any convenient reaction vessel, provided that the two processes are linked such that the C1 compounds from the reaction of H2 and CO2 are used in the fermentation as the starting product of the fermentation.

[0147] It should be noted that the embodiments and features described with respect to one embodiment of the invention also apply to other aspects of the invention.

[0148] All patent and non-patent literature cited in this application is hereby incorporated by reference in its entirety.

Claims

1. A method for producing single cell protein, the method comprising the following steps: (a) providing gaseous hydrogen (H ２ ); (b) Mixing the hydrogen gas from step (a) with a carbon source and carrying out either (i) a cell-free reaction of H2 with a first carbon source or (ii) a fermentation process to provide a C1 compound such as methane, methanol, formaldehyde, formic acid, methanethiol, or methanesulfonic acid, or a derivative thereof; (c) Adding or passing the C1 compound provided in step (b) to a fermentation reactor containing one or more microorganisms capable of metabolizing the C1 compound to provide an inoculated fermentation medium; (d) Fermenting the inoculated fermentation medium in a fermentation process to convert the C1 compound into biomass material; and (e) Isolating the biomass material provided in step (d) to provide single cell protein, Here, the carbon source is gaseous carbon monoxide (CO); gaseous carbon dioxide (CO ２ ); or a combination thereof, or the first carbon source is an aqueous solution of carbon dioxide such as carbonic acid, bicarbonate ions or carbonate ions or a combination thereof, and when step (b)(ii) is carried out, all steps are carried out in the same fermentation reactor.

2. The method according to claim 1, wherein the cell-free reaction is a Sabatier reaction or a reaction of CO2 to formic acid catalyzed by formate dehydrogenase or carbon dioxide reductase.

3. The CO and / or CO produced in the fermentation process ２ is returned to the fermentation process defined in step b, the method according to claim 1 or 2.

4. The method according to claim 1, wherein all steps are carried out in the same fermentation reactor.

5. The method according to claim 4, wherein the same fermentation reactor includes different compartments or zones for carrying out steps b and d.

6. The method according to claim 4 or 5, wherein the same fermentation reactor is run in a cycle including at least one cycle of running step b and at least one cycle of running step d.

7. H ２ The method according to claim 1, wherein H is a product of electrolysis or a product from a natural gas deposit.

8. The method according to claim 7, wherein the electrolysis is the electrolysis of natural water, such as seawater, using an electric current generated from a sustainable energy source such as wind power, wave power, tidal power, sunlight, geothermal energy, and hydropower.

9. The method according to claim 1, wherein the fermentation reactor is a loop reactor.

10. The method according to claim 9, wherein the loop reactor is a horizontal loop reactor.

11. The method according to claim 9, wherein the loop reactor is a vertical loop reactor.

12. The method according to claim 9, wherein the loop reactor is an inclined loop reactor.