Biomass production

Abstract

This invention generally relates to methods and systems for producing biomass from an input gas. This invention also generally relates to methods and systems for decreasing the amount of certain gases present in an input gas, in particular methane and / or carbon dioxide. The methods and systems described herein involve culturing and / or coculturing one or more strains of methanotrophic bacteria, and one or more strains of phototroph.

Description

BIOMASS PRODUCTIONFIELD OF THE INVENTION

[0001] This invention generally relates to methods and systems for producing biomass from an input gas. This invention also generally relates to methods and systems for decreasing the amount of certain gases present in an input gas, in particular methane and / or carbon dioxide. In particular, the invention relates to culturing and / or coculturing one or more strains of thermophilic, acidophilic, or thermoacidophilic methanotrophic bacteria, and one or more strains of thermophilic, acidophilic, or thermoacidophilic phototroph, such as microalgae.BACKGROUND TO THE INVENTION

[0002] Climate change is one of the major challenges facing humanity today. The major driver of climate change is the accumulation of greenhouse gases in the atmosphere that trap the sun's heat and increase temperature.

[0003] Methane and carbon dioxide are potent greenhouse gases and significant byproducts of industrial processes such as geothermal power generation, and oil and gas production. While methane is a relatively low proportion of anthropogenic greenhouse gas emissions, it is nevertheless a significant contributor to climate change, as it absorbs infrared radiation much more efficiently than carbon dioxide and has a warming potential ~86 times larger than its mass equivalent of carbon dioxide on a 20-year timescale (IPCC, 2014, 'Mitigation of Climate Change', Contribution of Working Group III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change, Cambridge University Press, Cambridge).

[0004] Carbon dioxide and methane can be useful in certain industrial processes, however these gases are often produced at concentrations too dilute for traditional economic utility, and are commonly released into the atmosphere via flaring or venting. The release of carbon dioxide and methane into the atmosphere therefore represents not only a cost in terms of greenhouse gas emissions, but also a lost opportunity to make use of these gases.

[0005] It is therefore desirable to provide methods and / or systems for decreasing the methane and / or carbon dioxide content of an input gas. It is also desirable to provide methods and / or systems for producing biomass from an input gas.

[0006] It is an object of the invention to go some way towards meeting one or more of these desiderata, or at least to provide the public with a useful choice.

[0007] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.SUMMARY OF THE INVENTION

[0008] In a first aspect, the invention provides a method for producing biomass from an input gas, the method comprising: a) coculturing one or more strains of methanotrophic bacteria and one or more strains of phototroph in the presence of the input gas and light to produce an output gas, or b) culturing one or more strains of methanotrophic bacteria in the presence of the input gas to produce an intermediate gas, and culturing one or more strains of phototroph in the presence of the intermediate gas and light to produce an output gas, or c) culturing one or more strains of phototroph in the presence of the input gas and light to produce an intermediate gas, and culturing one or more strains of methanotrophic bacteria in the presence of the intermediate gas to produce an output gas; wherein the one or more strains of methanotrophic bacteria and the one or more strains of phototroph are thermophilic, acidophilic, or thermoacidophilic; and wherein the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph produce biomass.

[0009] In a second aspect, the invention provides a method for decreasing the carbon containing gas content (for example, the methane and / or carbon dioxide content) of an input gas, the method comprising: a) coculturing one or more strains of methanotrophic bacteria and one or more strains of phototroph in the presence of the input gas and light to produce an output gas, or b) culturing one or more strains of methanotrophic bacteria in the presence of the input gas to produce an intermediate gas, and culturing one or morestrains of phototroph in the presence of the intermediate gas and light to produce an output gas, or c) culturing one or more strains of phototroph in the presence of the input gas and light to produce an intermediate gas, and culturing one or more strains of methanotrophic bacteria in the presence of the intermediate gas to produce an output gas; wherein the one or more strains of methanotrophic bacteria and the one or more strains of phototroph are thermophilic, acidophilic, or thermoacidophilic; and wherein the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph remove at least a portion of the carbon containing gas from the input gas and / or the intermediate gas.

[0010] In a third aspect, the invention provides a system for producing biomass from an input gas, the system comprising one or more strains of methanotrophic bacteria and one or more strains of phototroph, wherein the system is adapted to receive the input gas and to: a) coculture the one or more strains of methanotrophic bacteria and the one or more strains of phototroph in the presence of the input gas and light to produce an output gas, or b) culture the one or more strains of methanotrophic bacteria in the presence of the input gas to produce an intermediate gas, and culture the one or more strains of phototroph in the presence of the intermediate gas and light to produce an output gas, or c) culture the one or more strains of phototroph in the presence of the input gas and light to produce an intermediate gas, and culture the one or more strains of methanotrophic bacteria in the presence of the intermediate gas to produce an output gas; wherein the one or more strains of methanotrophic bacteria and the one or more strains of phototroph are thermophilic, acidophilic, or thermoacidophilic; and wherein, when in use, at least one of the one or more strains of methanotrophic bacteria and / or at least one of the one or more strains of phototroph produce biomass.

[0011] In a fourth aspect, the invention provides a system for decreasing the carbon containing gas content (for example, the methane and / or carbon dioxide content) of an inputgas, the system comprising one or more strains of methanotrophic bacteria and one or more strains of phototroph, wherein the system is adapted to receive the input gas and to: a) coculture the one or more strains of methanotrophic bacteria and the one or more strains of phototroph in the presence of the input gas and light to produce an output gas, or b) culture the one or more strains of methanotrophic bacteria in the presence of the input gas to produce an intermediate gas, and culture the one or more strains of phototroph in the presence of the intermediate gas and light to produce an output gas, or c) culture the one or more strains of phototroph in the presence of the input gas and light to produce an intermediate gas, and culture the one or more strains of methanotrophic bacteria in the presence of the intermediate gas to produce an output gas; wherein the one or more strains of methanotrophic bacteria and the one or more strains of phototroph are thermophilic, acidophilic, or thermoacidophilic; and wherein, when in use, at least one of the one or more strains of methanotrophic bacteria and / or at least one of the one or more strains of phototroph remove at least a portion of the carbon containing gas from the input gas and / or the intermediate gas.

[0012] In a fifth aspect, the invention provides biomass produced by the method or using the system of any previous aspect.

[0013] In a sixth aspect, the invention provides biomass comprising, or produced by, a coculture of Methylacidiphilum and Galdieria, preferably Methylacidiphilum sp. RTK17.1, KCTC accession number KCTC 15819BP dated 19 February 2024, or a derivative thereof, and Galdieria sp. RTK37.1, KCTC accession number KCTC 15846BP dated 7 March 2024, or a derivative thereof.

[0014] In a further aspect, the invention provides a nutritional composition comprising the biomass of the fifth or sixth aspects.

[0015] In a further aspect, the invention provides a fertiliser composition comprising the biomass of the fifth or sixth aspects.

[0016] Any of the following embodiments, alone or in any combination, may apply to any one or more of the above aspects.

[0017] In some embodiments, the method further comprises the step of recirculating at least a portion of the output gas such that the culturing one or more strains of methanotrophic bacteria, the culturing one or more strains of phototroph, and / or the coculturing, is in the presence of the input and recirculated gases.

[0018] In some embodiments, the system is adapted to recirculate at least a portion of the output gas, and wherein the system is adapted to culture and / or coculture the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph in the presence of the input and recirculated gases.

[0019] In some embodiments, the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph are thermoacidophilic.

[0020] In some embodiments, the one or more strains of methanotrophic bacteria comprise, or consist of, a strain from the phylum Verrucomicrobiota, preferably from the order Methyladdiphilal.es, more preferably from the family Methyladdiphilaceae, more preferably from the genus Methyladdiphilum, Methyladdimicrobium, or Methyladdithermus, more preferably from the genus Methyladdiphilum, most preferably from the strain Methyladdiphilum sp. RTK17.1, KCTC accession number KCTC 15819BP dated 19 February 2024, or a derivative thereof. In some embodiments, the one or more strains of methanotrophic bacteria have a 16S rRNA gene sequence that comprises a sequence with at least about 70% sequence identity to SEQ ID No: 1, such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID No: 1.

[0021] In some embodiments, the one or more strains of methanotrophic bacteria belong to the genus Methyladdiphilum, Methyladdithermus, and / or Methyladdimicrobium.

[0022] In some embodiments, the one or more strains of methanotrophic bacteria are selected from the group consisting of Methyladdiphilum sp. RTK17.1 KCTC accession number KCTC 15819BP dated 19 February 2024, or a derivative thereof, Methyladdiphilum fumariolicum, Methyladdiphilum infernorum, Methyladdiphilum kamchatkense, Methyladdiphilum caldifontis Methyladdiphilum sp. Yel, Methyladdiphilum sp. Phi, Methyladdithermus pantelleria, Methyladdithermus RTK, Methyladdimicrobium cyclophantes, Methyladdimicrobium fagopyrum, Methyladdimicrobium tartarophylax, and Methyladdimicrobium sp. LP2A. Preferably, the one or more strains of methanotrophic bacteria are selected from the group consisting of Methyladdiphilum sp. RTK17.1 KCTC accession number KCTC 15819BP dated 19 February 2024, or a derivative thereof, Methyladdiphilum kamchatkense, and Methyladdiphilum caldifontis.

[0023] In some embodiments, the one or more strains of phototroph comprise, or consist of, a strain from the order Cyan id idles, preferably from the family Cyanidiaceae or Galdieriaceae, more preferably from the genera Cyanidioschyzon, Cyanidium, Galdieria or Cyanidiococcus, more preferably from the genus Galdieria, most preferably Galdieria sp. RTK37.1 KCTC accession number KCTC 15846BP dated 7 March 2024, or a derivative thereof. In some embodiments, the one or more strains of phototroph have an 18S rRNA gene sequence that comprises a sequence with at least about 60% sequence identity to SEQ ID No: 2, such as at least about 75%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID No: 2.

[0024] In some embodiments, the one or more strains of phototroph belong to the genus Galdieria, Cyanidioschyzon, Cyanidiococcus, and / or Cyanidium.

[0025] In some embodiments, the one or more strains of phototroph are selected from the group consisting of Galdieria sp. RTK37.1 KCTC accession number KCTC 15846BP dated 7 March 2024, or a derivative thereof, Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria phlegrea, Galdieria sulphuraria, Cyanidioschyzon RTK, Cyanidioschyzon merolae, Cyanidiococcus vangmingshanesis, Cyanidium RTK, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, and Cyanidium rumpens. Preferably, the one or more strains of phototroph are selected from the group consisting of Galdieria sp. RTK37.1 KCTC accession number KCTC 15846BP dated 7 March 2024, or a derivative thereof, Galdieria sulphuraria, and Cyanidium caldarium.

[0026] In some embodiments, the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph are able to grow at, or have optimal growth at, a temperature of at least about 40°C, preferably at least about 45°C, more preferably from about 45°C to about 55°C. In some embodiments, culturing the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph comprises incubating at a temperature of at least about 40°C, preferably at least 45°C, more preferably from about 45°C to about 55°C.

[0027] In some embodiments the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph are able to grow at, or have optimal growth at, a pH of less than about 4.0, preferably less than about 3.0, more preferably from about 1.8 to about 2.5. In some embodiments, culturing the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph comprises culturing in a medium having a pH of less than about 4.0, preferably less than about 3.0, more preferably having a pH of from about 1.8 to about 2.5.

[0028] In some embodiments, the light comprises sunlight and / or light provided by one or more light-emitting devices. In some embodiments, the light is at an intensity of at least about 30 pmol photons m’2s’1, preferably at least about 100 pmol photons m’2s’1.

[0029] In some embodiments, the culturing and / or coculturing takes place at a pressure above atmospheric pressure, preferably at least about 100 mbar above atmospheric pressure, more preferably at least about 500 mbar above atmospheric pressure.

[0030] In some embodiments, the initial dry weight mass ratio of the one or more strains of methanotrophic bacteria to the one or more strains of phototroph is less than about 0.5, preferably less than about 0.3, more preferably between about 0.1 and about 0.3, most preferably about 0.15.

[0031] In some embodiments, the input gas comprises methane. In some embodiments, the input gas comprises at least about 0.1% v / v methane, such as at least about 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or at least about 20% v / v methane, and useful ranges may be selected between any of these values (for example, from about 0.1% to about 20%, from about 0.1% to about 15%, from about 0.1% to about 10%, from about 0.1% to about 5%, from about 0.1% to about 4%, from about 0.1% to about 3%, from about 0.1% to about 2%, from about 0.1% to about 1%, from about 0.5% to about 20%, from about 0.5% to about 15%, from about 0.5% to about 10%, from about 0.5% to about 5%, from about 0.5% to about 4%, from about 0.5% to about 3%, from about 0.5% to about 2%, from about 0.5% to about 1%, from about 1.0% to about 20%, from about 1.0% to about 15%, from about 1.0% to about 10%, from about 1.0% to about 5%, from about 1.0% to about 4%, from about 1.0% to about 3%, from about 1.0% to about 2%, or from about 1.0% to about 1.5% v / v).

[0032] In some embodiments, the input gas comprises oxygen. In some embodiments, the input gas comprises at least about 1.0% v / v oxygen, such as at least about 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or at least about 20% v / v oxygen, and useful ranges may be selected between any of these values (for example, from about 1.0% to about 20%, from about 1.0% to about 15%, from about 1.0% to about 10%, from about 1.0% to about 5%, from about 1.0% to about 4%, from about 1.0% to about 3%, from about 1.0% to about 2.5%, from about 1.0% to about 2.2%, from about 1.5% to about 20%, from about 1.5% to about 15%, from about 1 .5% to about 10%, from about 1.5% to about 5%, from about 1.5% to about 4%, from about 1.5% to about3%, from about 1.5% to about 2.5%, or from about 1.5% to about 2.2%). Preferably, the input gas comprises about 3% v / v oxygen.

[0033] In some embodiments, the input gas comprises carbon dioxide. In some embodiments, the input gas comprises at least about 10% v / v carbon dioxide, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or at least about 98% v / v carbon dioxide, and useful ranges may be selected between any of these values (for example, from about 10% to about 98%, from about 10% to about 94%, from about 10% to about 90%, from about 10% to about 86%, from about 10% to about 80%, from about 10% to about 75%, from about 10% to about 70%, from about 10% to about 65%, from about 10% to about 60%, from about 20% to about 98%, from about 20% to about 94%, from about 20% to about 90%, from about 20% to about 86%, from about 20% to about 80%, from about 20% to about 75%, from about 20% to about 70%, from about 20% to about 65%, from about 20% to about 60%, from about 30% to about 98%, from about 30% to about 94%, from about 30% to about 90%, from about 30% to about 86%, from about 30% to about 80%, from about 30% to about 75%, from about 30% to about 70%, from about 30% to about 65%, from about 30% to about 60%, from about 40% to about 98%, from about 40% to about 94%, from about 40% to about 90%, from about 40% to about 86%, from about 40% to about 80%, from about 40% to about 75%, from about 40% to about 70%, from about 40% to about 65%, from about 40% to about 60%, from about 50% to about 98%, from about 50% to about 94%, from about 50% to about 90%, from about 50% to about 86%, from about 50% to about 80%, from about 50% to about 75%, from about 50% to about 70%, from about 50% to about 65%, or from about 50% to about 60% v / v). Preferably, the input gas comprises about 73% v / v carbon dioxide.

[0034] In some embodiments, the input gas comprises from about 1.0% to about 15% v / v methane, from about 1.0% to about 20% v / v oxygen, and / or from about 10% to about 98% v / v carbon dioxide. In some embodiments, the input gas, intermediate gas, and / or output gas is not an explosive mixture.

[0035] In some embodiments, the input gas comprises H2S. In some embodiments the input gas comprises at least about 100 ppm H2S, such as at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or at least about 6000 ppm H2S, and useful ranges may be selected between any of these values (for example, from about 100 to about 6000, from about 100 to about 5000, from about 100 to about 4000, from about 100 to about 3000, from about 100 to about 2000, from about 100 to about 1000, from about 100 to about 800, from about 100 to about 600, from about 300 to about 6000, from about 300 to about 5000, from about 300 to about4000, from about 300 to about 3000, from about 300 to about 2000, from about 300 to about 1000, from about 300 to about 800, or from about 300 to about 600).

[0036] In some embodiments, the culturing and / or coculturing occurs in a culture medium, preferably an aqueous culture medium. In some embodiments, the culture medium has a pH of less than about 4.0, preferably less than about 3.0, more preferably from about 1.8 to about 2.5. In some embodiments, the culture medium comprises at least about 0.2 mg / L of dissolved oxygen, preferably at least about 1 mg / L of dissolved oxygen.

[0037] In some embodiments, the input gas is at a temperature of at least about 35°C, such as at least about 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or at least about 100°C, and useful ranges may be selected between any of these values (for example, from about 35°C to about 100°C, from about 35°C to about 90°C, from about 35°C to about 80°C, from about 35°C to about 70°C, from about 40°C to about 100°C, from about 40°C to about 90°C, from about 40°C to about 80°C, from about 40°C to about 70°C, from about 45°C to about 100°C, from about 45°C to about 90°C, from about 45°C to about 80°C, from about 45°C to about 70°C, from about 50°C to about 100°C, from about 50°C to about 90°C, from about 50°C to about 80°C, from about 50°C to about 70°C, from about 55°C to about 100°C, from about 55°C to about 90°C, from about 55°C to about 80°C, from about 55°C to about 70°C, from about 60°C to about 100°C, from about 60°C to about 90°C, from about 60°C to about 80°C, from about 60°C to about 70°C).

[0038] In some embodiments, the input gas comprises a waste gas stream from one or more industrial processes, preferably from power generation (such as geothermal power generation), oil production, and / or natural gas production. In some embodiments, the input gas comprises gas produced by the microbial degradation of organic material.

[0039] In some embodiments, the output gas has a reduced content of carbon containing gas (for example, methane and / or carbon dioxide) compared to the input gas. In some embodiments, the output gas has a carbon containing gas content (for example, methane and / or carbon dioxide content) that is less than about 50% of that of the input gas.

[0040] In some embodiments, the output gas contains less than about 10% v / v methane, preferably less than about 1% v / v, more preferably less than about 0.1% v / v.

[0041] In some embodiments, the biomass comprises: a) one or more biopolymers, preferably one or more proteins, glycoproteins, polypeptides, polysaccharides, and / or polynucleotides, b) one or more amino acids,c) one or more nucleotides, and / or nucleosides, d) one or more carbohydrates, e) one or more lipids, f) one or more vitamins, g) one or more bioactive compounds, preferably one or more pigment-containing compounds, more preferably phycocyanin, or h) any combination of any two or more of (a) to (g).

[0042] In some embodiments, the biomass comprises at least about 20% w / w protein by dry weight, preferably at least about 30%, more preferably at least about 40%, most preferably at least about 50% w / w protein by dry weight.

[0043] In some embodiments, the biomass has an essential amino acid concentration of at least about 6 g / 100g dry weight, preferably at least about 10 g / 100g dry weight, more preferably at least about 18 g / 100g dry weight.

[0044] In some embodiments, the biomass comprises: a) at least about 2.5 g lysine per 100 g protein, preferably at least about 4.5 g per 100 g protein; b) at least about 1.0 g methionine per 100 g protein, preferably at least about 1.6 g per 100 g protein; c) at least about 0.4 g cysteine per 100 g protein, preferably at least about 0.6 g per 100 g protein; or d) any combination of any two or more of (a) to (c).

[0045] In some embodiments, the biomass comprises protein that, if consumed as the sole dietary protein source at 0.66 g protein kg-1day-1, would meet or exceed the required daily human intake for: a) isoleucine, b) leucine, c) lysine, d) methionine,e) phenylalanine, f) threonine, g) tryptophan, h) valine, or i) any combination of any two or more of (a) to (h).

[0046] In some embodiments, the method further comprises the step of recovering biomass from the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph.

[0047] In some embodiments, the biomass comprises protein. In some embodiments, the biomass comprises at least about 1% w / w protein by dry weight, such as at least about 5%, 10%, 15%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 68%, or at least about 70% w / w protein by dry weight, and useful ranges may be selected between any of these values (for example, from about 1% to about 70%, from about 1% to about 60%, from about 1% to about 40%, from about 1% to about 20%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 40%, from about 10% to about 20%, from about 20% to about 70%, from about 20% to about 60%, from about 20% to about 58%, from about 20% to about 56%, from about 20% to about 54%, from about 20% to about 52%, from about 20% to about 50%, from about 30% to about 70%, from about 30% to about 60%, from about 30% to about 58%, from about 30% to about 56%, from about 30% to about 54%, from about 30% to about 52%, from about 30% to about 50%, from about 40% to about 70%, from about 40% to about 60%, from about 40% to about 58%, from about 40% to about 56%, from about 40% to about 54%, from about 40% to about 52%, from about 40% to about 50%, from about 50% to about 70%, or from about 50% to about 60%).

[0048] In some embodiments, the biomass has an essential amino acid concentration of at least about 6 g / 100g dry weight, such as at least about 8g / 100g dry weight, at least about 10 g / 100g dry weight, at least about 12 g / 100g dry weight, at least about 14 g / 100g dry weight, at least about 16 g / 100g dry weight, at least about 18 g / 100g dry weight, at least about 20 g / 100g dry weight, at least about 22 g / 100g dry weight, at least about 24 g / 100g dry weight, or at least about 26 g / 100g dry weight, and useful ranges may be selected between any of these values (for example, from about 6 to about 26 g / 100g dry weight, from about 6 to about 24 g / 100g dry weight, from about 6 to about 22 g / 100g dry weight, from about 6 to about 20 g / 100g dry weight, from about 8 to about 26 g / 100g dry weight, from about 8 to about 24 g / 100g dry weight, from about 8 to about 22 g / 100g dry weight, fromabout 8 to about 20 g / 100g dry weight, from about 10 to about 26 g / 100g dry weight, from about 10 to about 24 g / 100g dry weight, from about 10 to about 22 g / 100g dry weight, from about 10 to about 20 g / 100g dry weight, from about 12 to about 26 g / 100g dry weight, from about 12 to about 24 g / 100g dry weight, from about 12 to about 22 g / 100g dry weight, from about 12 to about 20 g / 100g dry weight, from about 14 to about 26 g / 100g dry weight, from about 14 to about 24 g / 100g dry weight, from about 14 to about 22 g / 100g dry weight, from about 14 to about 20 g / 100g dry weight, from about 16 to about 26 g / 100g dry weight, from about 16 to about 24 g / 100g dry weight, from about 16 to about 22 g / 100g dry weight, from about 16 to about 20 g / 100g dry weight, from about 18 to about 26 g / 100g dry weight, from about 18 to about 24 g / 100g dry weight, from about 18 to about 22 g / 100g dry weight, or from about 18 to about 20 g / 100g dry weight).

[0049] In some embodiments, the biomass has an essential amino acid concentration of at least about 20 g / 100g protein, such as at least about 25 g / 100g protein, at least about 30 g / 100g protein, at least about 35 g / 100g protein, or at least about 40 g / 100g protein, and useful ranges may be selected between any of these values (for example, from about 20 to about 40 g / 100g protein, from about 20 to about 35 g / 100g protein, from about 25 to about 40 g / 100g protein, from about 25 to about 35 g / 100g protein, from about 30 to about 40 g / 100g protein, or from about 30 to about 35 g / 100g protein).

[0050] In some embodiments, the biomass comprises at least about 2.5 g lysine per 100 g protein, such as at least about 3.0, 3.5, 4.0, 4.5, 5.0, or at least about 5.5 g lysine per 100 g protein.

[0051] In some embodiments, the biomass comprises at least about 1.0 g methionine per 100 g protein, such as at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, or at least about 2.2 g methionine per 100 g protein.

[0052] In some embodiments, the biomass comprises at least about 0.4 g cysteine per 100 g protein, such as at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, or at least about 1.7 g cysteine per 100 g protein.

[0053] In some embodiments, the biomass comprises fat. In some embodiments, the biomass comprises at least about 1% w / w fat by dry weight, such as at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or at least about 10% w / w fat by dry weight, and useful ranges may be selected between any of these values (for example, from about 1% to about 10%, from about 1% to about 9%, from about 1% to about 8%, from about 1% to about 7%, from about 1% to about 6%, from about 1% to about 5%, from about 2% to about 10%, from about 2% to about 9%, from about 2% to about 8%, from about 2% to about 7%, from about 2% to about 6%, from about 2% to about 5%, from about 3% to about 10%, from about 3% toabout 9%, from about 3% to about 8%, from about 3% to about 7%, from about 3% to about 6%, from about 3% to about 5%, from about 4% to about 10%, from about 4% to about 9%, from about 4% to about 8%, from about 4% to about 7%, from about 4% to about 6%, or from about 4% to about 5%).

[0054] In some embodiments, the biomass comprises carbohydrate. In some embodiments, the biomass comprises at least about 10% w / w carbohydrate by dry weight, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or at least about 70% w / w carbohydrate by dry weight, and useful ranges may be selected between any of these values (for example, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 20% to about 70%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 30% to about 70%, from about 30% to about 60%, from about 30% to about 50%, or from about 30% to about 40%).

[0055] In some embodiments, the nutritional composition is a human nutritional composition. In some embodiments, the human nutritional composition is a food, a beverage, a food ingredient, or a beverage ingredient.

[0056] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the animal feed composition is a livestock food, a pet food, a kibble, pet biscuit, a pet treat, an animal feed ingredient, a livestock forage, a livestock supplement, or a drench.

[0057] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0058] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0059] Although the present invention is broadly as defined above, those persons skilled in the art will appreciate that the invention is not limited thereto and that the invention also includes embodiments of which the following description gives examples.

[0060] In the description in this specification reference may be made to subject matter which is not within the scope of the claims of the current application. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this application.Brief description of the drawings

[0061] The invention will now be described by way of example only and with reference to the drawings in which:

[0062] Figure 1 shows the concentration profile over time of compounds in Galdieria sp. RTK37.1 culture (circles), Methylacidiphilum sp. RTK17.1 culture (squares), and cocultures (triangles), (a) O2 headspace concentration, (b) CH4 headspace concentration, (c) CO2 headspace concentration, (d) NH4+concentration. Values shown are the average of duplicate experiments, with error bars representing one standard deviation.

[0063] Figure 2 shows production and consumption rates of Galdieria sp. RTK37.1 culture (circles), Methylacidiphilum sp. RTK17.1 culture (squares), and cocultures (triangles), (a) O2 net production rates, (b) CH4 consumption rates, (c) CO2 net production rates, (d) NH4+consumption rates. Values shown are the average of duplicate experiments, with error bars representing one standard deviation.

[0064] Figure 3 shows the indispensable amino acid composition of cultures and cocultures of Methylacidiphilum sp. RTK17.1, Galdieria sp. RTK37.1, and reference proteins. Error bars represent standard deviations with n=3 technical replicates, except for RTK37.1 (chemostat) with n=2. The horizontal dashed line represents the FAO / WHO / UNU reference protein.

[0065] Figure 4 shows the effect of initial Methylacidiphilum:Galdieria ratios on coculture growth with (triangles) and without (circles) CH4 supplementation into the headspace, (a) average growth rate over the entire period, (b) biomass productivity.

[0066] Figure 5 shows the effect of initial Methylacidiphilum:Galdieria ratios on coculture growth with (triangles) and without (circles) CH4 supplementation into the headspace, (a) net CO2 consumption, (b) net CH4 consumption, (c) net C fixation, (d) net O2 production.

[0067] Figure 6 shows the effect of O2 inlet concentration on steady state biomass concentration for cultures of Methylacidiphilum sp. RTK17.1 (circles), Galdieria sp. RTK37.1 (triangles), and cocultures (squares). Values shown are the average of triplicate measurements, with error bars representing one standard deviation.

[0068] Figure 7 shows the steady state biomass concentrations of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 at different O2 concentrations in chemostat cocultures and axenic controls, (a) Methylacidiphilum concentration in axenic culture (circles and solid line) and in coculture (squares and dashed line), (b) Galdieria concentration in axenic culture (triangles and solid line) and in coculture (squares and dashed line). Values shown are an average of five measurements, with error bars representing one standard deviation.

[0069] Figure 8 shows the fluorescence of (a) chlorophyl, (b) phycocyanin, and (c) coproporphyrin in axenic Galdieria sp. RTK37.1 cultures (triangles) and in cocultures (squares). Values shown are the average of five measurements, with error bars representing one standard deviation.

[0070] Figure 9 shows steady state gas for axenic Methylacidiphilum sp. RTK17.1 cultures (circles), axenic Galdieria sp. RTK37.1 cultures (triangles) and co-cultures (squares), (a) Dissolved O2 concentration, (b) Net O2 production rate, (c) O2 specific net rate, (d) Net CH4 consumption rate, (e) CH4 specific consumption rate, with rate calculated using total biomass shown as filled squares, and the rate calculated using Methylacidiphilum biomass only as unfilled squares.

[0071] Figure 10 shows the concentration profile of a high density coculture of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1. (a) ODeoo of the coculture (circles) and relative abundance of Methylacidiphilum (squares) and Galdieria (triangles), (b) Total biomass concentration measured gravimetrically. Dashed vertical lines indicate the addition of ammonium chloride to the reactor, and shading indicates the light intensity used: 100 (left, dark grey), 150 (middle, light grey) and 200 (right, white) nmol photons m’2s’1.DETAILED DESCRIPTION OF THE INVENTION

[0072] The inventors have found that cultures and cocultures of methanotrophic bacteria and phototrophs are surprisingly effective at removing unwanted gases such as methane and carbon dioxide from an input gas. The inventors have also found that such cocultures are capable of producing biomass that may be useful for a variety of purposes, for example as a component of nutritional compositions.

[0073] Without wishing to be bound by theory, it is believed that uni- or bi-directional cross-feeding between methanotrophic bacteria and phototrophs may result in improvedgrowth of one or more of the microorganisms, improved removal of the unwanted gases, and / or greater biomass production. For example, the phototroph may remove carbon dioxide and produce oxygen and organic metabolites by oxygenic photosynthesis, while the methanotrophic bacteria may utilise this oxygen to remove methane and produce carbon dioxide via respiration.

[0074] The inventors have also found that the use of extremophiles such as thermophilic, acidophilic, or preferably thermoacidophilic microorganisms has particular benefits. For example, the use of such microorganisms allows the culturing and / or coculturing to occur at higher temperatures and / or lower pH than non-extremophile microorganisms. This can result in a decreased need for cooling of an input gas, and also provides resistance to contamination by unwanted microorganisms, as most such contaminating microorganisms are unable to grow, or have reduced growth rate, at these temperatures and / or pHs.

[0075] Accordingly, in a first aspect, the invention provides a method for producing biomass from an input gas, the method comprising: a) coculturing one or more strains of methanotrophic bacteria and one or more strains of phototroph in the presence of the input gas and light to produce an output gas, or b) culturing one or more strains of methanotrophic bacteria in the presence of the input gas to produce an intermediate gas, and culturing one or more strains of phototroph in the presence of the intermediate gas and light to produce an output gas, or c) culturing one or more strains of phototroph in the presence of the input gas and light to produce an intermediate gas, and culturing one or more strains of methanotrophic bacteria in the presence of the intermediate gas to produce an output gas; wherein the one or more strains of methanotrophic bacteria and the one or more strains of phototroph are thermophilic, acidophilic, or thermoacidophilic; and wherein the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph produce biomass.

[0076] In a second aspect, the invention provides a method for decreasing the carbon containing gas content (for example, the methane and / or carbon dioxide content) of an input gas, the method comprising:a) coculturing one or more strains of methanotrophic bacteria and one or more strains of phototroph in the presence of the input gas and light to produce an output gas, or b) culturing one or more strains of methanotrophic bacteria in the presence of the input gas to produce an intermediate gas, and culturing one or more strains of phototroph in the presence of the intermediate gas and light to produce an output gas, or c) culturing one or more strains of phototroph in the presence of the input gas and light to produce an intermediate gas, and culturing one or more strains of methanotrophic bacteria in the presence of the intermediate gas to produce an output gas; wherein the one or more strains of methanotrophic bacteria and the one or more strains of phototroph are thermophilic, acidophilic, or thermoacidophilic; and wherein the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph remove at least a portion of the carbon containing gas from the input gas and / or the intermediate gas.

[0077] In a third aspect, the invention provides a system for producing biomass from an input gas, the system comprising one or more strains of methanotrophic bacteria and one or more strains of phototroph, wherein the system is adapted to receive the input gas and to: a) coculture the one or more strains of methanotrophic bacteria and the one or more strains of phototroph in the presence of the input gas and light to produce an output gas, or b) culture the one or more strains of methanotrophic bacteria in the presence of the input gas to produce an intermediate gas, and culture the one or more strains of phototroph in the presence of the intermediate gas and light to produce an output gas, or c) culture the one or more strains of phototroph in the presence of the input gas and light to produce an intermediate gas, and culture the one or more strains of methanotrophic bacteria in the presence of the intermediate gas to produce an output gas; wherein the one or more strains of methanotrophic bacteria and the one or more strains of phototroph are thermophilic, acidophilic, or thermoacidophilic; andwherein, when in use, at least one of the one or more strains of methanotrophic bacteria and / or at least one of the one or more strains of phototroph produce biomass.

[0078] In a fourth aspect, the invention provides a system for decreasing the carbon containing gas content (for example, the methane and / or carbon dioxide content) of an input gas, the system comprising one or more strains of methanotrophic bacteria and one or more strains of phototroph, wherein the system is adapted to receive the input gas and to: a) coculture the one or more strains of methanotrophic bacteria and the one or more strains of phototroph in the presence of the input gas and light to produce an output gas, or b) culture the one or more strains of methanotrophic bacteria in the presence of the input gas to produce an intermediate gas, and culture the one or more strains of phototroph in the presence of the intermediate gas and light to produce an output gas, or c) culture the one or more strains of phototroph in the presence of the input gas and light to produce an intermediate gas, and culture the one or more strains of methanotrophic bacteria in the presence of the intermediate gas to produce an output gas; wherein the one or more strains of methanotrophic bacteria and the one or more strains of phototroph are thermophilic, acidophilic, or thermoacidophilic; and wherein, when in use, at least one of the one or more strains of methanotrophic bacteria and / or at least one of the one or more strains of phototroph remove at least a portion of the carbon containing gas from the input gas and / or the intermediate gas.1. Definitions

[0079] Unless otherwise stated, the singular forms "a", "an" and "the" include the plural reference.

[0080] The term "about" as used herein generally refers to a range of numerical values (e.g. ± 5 to 10% of the recited value) that those skilled in the art would consider equivalent to the recited value. Ranges can be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, the range is inclusive of the recited values.

[0081] The term "and / or" as used herein means "and" or "or", or both.

[0082] The term "(s)" following a noun as used herein means the plural and / or singular form of that noun.

[0083] The term "acidophilic" as used herein means a microorganism that is able to grow at a pH of about 5.0 or less. In some embodiments, an acidophilic microorganism (such as an acidophilic methanotrophic bacterium, a thermoacidophilic methanotrophic bacterium, an acidophilic microalga, or a thermoacidophilic microalga) is able to grow at, or has optimal growth at, a pH of about 4.5 or less, such as about 4.0 or less, about 3.9 or less, about 3.8 or less, about 3.7 or less, about 3.6 or less, about 3.5 or less, about 3.4 or less, about 3.3 or less, about 3.2 or less, about 3.1 or less, about 3.0 or less, about 2.9 or less, about 2.8 or less, about 2.7 or less, about 2.6 or less, about 2.5 or less, about 2.4 or less, about 2.3 or less, about 2.2 or less, about 2.1 or less, about 2.0 or less, about 1.9 or less, about 1.8 or less, about 1.7 or less, about 1.6 or less, about 1.5 or less, about 1.4 or less, about 1.3 or less, about 1.2 or less, about 1.1 or less, about 1.0 or less, about 0.9 or less, about 0.8 or less, about 0.7 or less, about 0.6 or less, about 0.5 or less, about 0.4 or less, about 0.3 or less, about 0.2 or less, about 0.1 or less, or about 0.0 or less, and useful ranges may be selected between any of these values (for example, from about 0.0 to about 4.5, from about 0.0 to about 4.0, from about 0.0 to about 3.5, from about 0.0 to about 3.0, from about 0.0 to about 2.5, from about 0.5 to about 4.5, from about 0.5 to about 4.0, from about 0.5 to about 3.5, from about 0.5 to about 3.0, from about 0.5 to about 2.5, from about 1.0 to about 4.5, from about 1.0 to about 4.0, from about 1.0 to about 3.5, from about 1.0 to about 3.0, from about 1.0 to about 2.5, from about 1.8 to about 4.5, from about 1.8 to about 4.0, from about 1.8 to about 3.5, from about 1.8 to about 3.0, or from about 1.8 to about 2.5).

[0084] The term "animal feed" as used herein means material(s) that are consumed by animals and contribute energy and / or nutrients to an animal's diet. Animal feed includes (but is not limited to) feed for livestock, aquatic animal feed, and pet food. Animal feeds typically include a number of different components that may be present in forms such as concentrate(s), premix(es), co-product(s), or pellets. In some embodiments, biomass produced using the methods and / or systems of the invention may be useful as a component of animal feed. Examples of feeds and other feed components include Partial or Total Mixed Ration (TMR), corn, soybean, forage, grain, distiller's grain, sprouted grain, legumes, vitamins, amino acids, minerals, fibre, fodder, grass, hay, straw, silage, kernel, leaves, meal, solubles, slurries, supplements, mash feed, meal, fruit pulp, vegetable pulp, fruit or vegetable pomace, citrus meal, wheat shorts, corn cob meal, and molasses, milk, milk powder, milk replacement, milk fortifier, colostrum, whey, whey powder, sucrose, maltodextrin, rice hulls and the like.

[0085] The term "bioactive compound" as used herein means any compound with biological activity, for example antioxidant and / or anti-inflammatory activity. Exemplary bioactive compounds include secondary metabolites such as those produced by microbes. One preferred class of bioactive compounds is pigment-containing compounds produced by phototrophs, such as phycocyanin.

[0086] The term "biomass" as used herein means organic material that is produced by, from, and / or comprises, living organism(s). In some embodiments, biomass comprises whole cells, such as whole bacterial and / or phototroph cells. In some embodiments, biomass comprises cell products and / or derivatives, such as but not limited to heat-killed, lysed, fractionated, pressure-killed, irradiated, or UV- or light-treated cells, and / or material derived from the cells including but not limited to cell wall compositions, cell lysates, lyophilised cells, cell metabolites, cell suspensions, cell-free culture supernatant, and the like. Preferably, the biomass comprises one or more proteins, one or more amino acids, one or more carbohydrates, one or more lipids, one or more vitamins, or any combination of any two or more of these.

[0087] The term "biopolymer" as used herein means a polymer produced from or biosynthesised by a living organism, such as a methanotrophic bacterium or a phototroph. Exemplary biopolymers include proteins, glycoproteins, polypeptides, polysaccharides, and polynucleotides.

[0088] The term "carbon containing gas" as used herein means a gas that contains at least one carbon atom in its molecular structure. Exemplary carbon containing gases include methane (ChU), carbon dioxide (CO2), and carbon monoxide (CO).

[0089] The term "coculture" and related terms such as "coculturing" and "cocultured" as used herein means culturing two or more different microorganisms (preferably at least one strain of methanotrophic bacteria and at least one strain of phototroph) in such a way that cross-feeding can occur. In some embodiments, coculturing two or more different microorganisms comprises culturing the two or more different microorganisms within the same culture vessel.

[0090] The term "comprising" as used in this specification and claims means "consisting at least in part of". When interpreting statements in this specification and claims which include the term "comprising", other features besides the features prefaced by this term in each statement can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in similar manner.

[0091] The term "cross-feeding" as used herein means the exchange of nutrients between microorganisms. Cross-feeding includes one-way cross-feeding, in which a first microorganism produces one or more substances that are utilised by a second microorganism, and bidirectional cross-feeding in which a first microorganism produces one or more substances that are utilised by a second microorganism, and the second microorganism produces one or more substances that are utilised by the first. Cross-feeding may also involve three or more different microorganisms.

[0092] The term "explosive mixture" as used herein means a mixture of at least one flammable gas (for example, methane) and at least one oxidising gas (for example, oxygen) in proportions, concentrations, and pressure such that they are capable of explosive reaction when exposed to an ignition source. Janes et al. provides experimental determination of the flammability / explosivity of methane / oxygen / carbon dioxide mixtures (2011, 'Experimental study of CH4 / O2 / CO2 mixtures flammability.' 1 lAIChE - 2011 AIChE Spring Meeting and 7th Global Congress on Process Safety, Conference Proceedings).

[0093] The term "nutritional composition" as used herein refers to any edible and / or drinkable product or ingredient. The term is intended to include products for human consumption and products for non-human consumption, such as an animal feed composition. The term is also intended to include products that must undergo further processing before being in a consumable form, for example a drink powder that is dissolved in a liquid to form a beverage. It will be appreciated that in some embodiments, the human nutritional composition will be an ingredient for incorporation into other products, such as a bakery ingredient.

[0094] The term "thermophilic" as used herein means a microorganism that is able to grow at a temperature of at least about 45°C. In some embodiments, a thermophilic microorganism (such as a thermophilic methanotrophic bacterium, a thermoacidophilic methanotrophic bacterium, a thermophilic phototroph, or a thermoacidophilic phototroph) is able to grow at a temperature of at least about 46°C, at least about 48°C, at least about 50°C, at least about 52°C, at least about 54°C, at least about 56°C, at least about 58°C, at least about 60°C, at least about 62°C, at least about 64°C, at least about 66°C, at least about 68°C, at least about 70°C, at least about 72°C, at least about 74°C, at least about 76°C, at least about 78°C, or at least about 80°C.

[0095] The term "thermoacidophilic" as used herein means a microorganism that is both thermophilic and acidophilic.

[0096] The terms "methanotrophic" and "methanotroph" as used herein mean a microorganism that is able to metabolise methane as the primary or sole source of carbonand chemical energy. Methanotrophs may be bacteria or archaea, and may be able to oxidise methane under aerobic or anaerobic conditions. In some embodiments the methanotroph is a bacterium able to oxidise methane under aerobic conditions, preferably belonging to the phylum Verrucomicrobiota, more preferably a species of Methylacidiphilum, most preferably Methylacidiphilum sp. RTK17.1 KCTC accession number KCTC 15819BP dated 19 February 2024, or a derivative thereof.

[0097] The terms "microalga" and "microalgae" as used herein means unicellular algae, and includes both unicellular algae that live singly, and those that form colonies.

[0098] The term "phototroph" as used herein means an organism that is capable of converting light into chemical energy. Preferred phototrophs are photoautotrophs that can perform photosynthesis. Phototrophs may include (but are not limited to) algae including microalgae, and cyanobacteria. In some embodiments, the phototroph is an alga, preferably a microalga, more preferably from the family Cyanidiophyceae, more preferably a species of Galdieria, or most preferably Galdieria sp. RTK37 KCTC accession number KCTC 15846BP dated 7 March 2024, or a derivative thereof.

[0099] The term "waste gas stream" as used herein means gas produced as a byproduct or waste product. In some embodiments, a waste gas stream is produced by an industrial process such as power generation, oil production, or natural gas production. In other embodiments, a waste gas stream is produced by a biological process, such as the breakdown of organic waste, for example in landfill or sewage treatment.2. Methanotrophic bacteria

[0100] Methanotrophs are bacteria capable of using methane as their sole source of carbon and energy. Most studied methanotrophs are members of the gamma- or alphaproteobacterial classes and are classified as Type I or Type II, respectively. Most described Type I and II methanotrophs are neutrophilic and mesophilic, with the most acidic previously reported pH values, that supported methanotrophic growth, between 4.2 and 5.0 in bacteria isolated from peat bogs (Op den Camp et al., 2009. Environmental, genomic and taxonomic perspectives on methanotrophic Verrucomicrobia. Environmental Microbiology Reports, 1 (5), 293-306).

[0101] Three independent studies reported the discovery of thermoacidophilic aerobic methane-oxidizing bacteria within the phylum Verrucomicrobiota (formerly Verrucomicrobia) in fumaroles, with temperatures as hot as 70°C and pH as acidic as 1.8, incorporated herein by reference (Dunfield et aL, 2007, 'Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia', Nature, 450(7171), 879-882. doi:10.1038 / nature06411; Islam et aL, 2008, 'Methane oxidation at 55 degrees C and pH 2 by a thermoacidophilic bacteriumbelonging to the Verrucomicrobia phylum', PNAS, 105(1), 300-304. doi:10.1073 / pnas.0704162105; Pol et al., 2007, 'Methanotrophy below pH 1 by a new Verrucomicrobia species', Nature, 450, 874. doi:10.1038 / nature06222). The identified species were subsequently phylogenetically clustered into a single genus, Methylacidiphilum.

[0102] Mesophilic acidophilic verrucomicrobial methanotrophs have also been isolated from cooler geothermal sites and clustered under the genus Methylacidimicrobium.

[0103] Verrucomicrobial methanotrophs have been identified in geothermal environments over a broad temperature range (23 - 80°C), but only in acidic conditions (pH 0.8 - 5.0). For the thermoacidophilic verrucomicrobial methanotrophs (Methylacidiphilum spp.) the reported optimum temperature for growth is 55 - 60 °C and the optimum pH is 2.0 - 3.5 (Op den Camp et al., 2009, Verrucomicrobial Methanotrophs. In M. G. Kalyuzhnaya & X.- H. Xing (Eds.), Methane Biocatalysis: Paving the Way to Sustainability (pp. 43-55). Cham: Springer International Publishing; Sharp et al., 2014, Humboldt's spa: microbial diversity is controlled by temperature in geothermal environments. The Isme Journal, 8(6), 1166-1174. doi:10.1038 / ismej.2013.237).

[0104] Despite the prevalence of work over the past 20 years investigating the usage of conventional (Type I and Type II) methanotrophs for the production of biofuels, ectoines, biopolymers, methanol and exopolysaccharides, no studies have explicitly reported on the potential industrial applications of verrucomicrobial methanotrophs.

[0105] Verrucomicrobial Methylacidiphilum species can be found in most acidic geothermal fields that produce methane. The strain Methylacidiphilum sp. RTK17.1 was isolated in the Rotokawa region in New Zealand, a geothermal area with acidic soils and high methane and carbon dioxide emissions. Methylacidiphilum sp. RTK17.1 grows optimally at pH 2.5 and a temperature of 50°C, with a Tmax of 60°C, is capable of oxidising methane, fixing carbon dioxide, fixing nitrogen under nitrogen limiting conditions, accumulating glycogen, and rapidly consuming hydrogen under microaerophilic conditions. Carbon dioxide fixation occurs in cultures supplied with hydrogen as the sole reductant and oxygen as the sole oxidant. Characterisation of Methylacidiphilum sp. RTK17.1 is discussed in Carere et al., and incorporated herein by reference (Carere et al., 2017, 'Hydrogen Oxidation Influences Glycogen Accumulation in a Verrucomicrobial Methanotroph', Front. Microbiol. 10:1873, doi: 10.3389 / fmicb.2019.01873).

[0106] Op den Camp et al. summarises the methane oxidation and carbon fixation of verrucomicrobial methanotrophs, and is incorporated herein by reference (Op den Camp et al., 2018, 'Verrucomicrobial Methanotrophs', In M. G. Kalyuzhnaya & X.-H. Xing (Eds.), Methane Biocatalysis: Paving the Way to Sustainability (pp. 43-55). Cham: SpringerInternational Publishing.). In brief, proteobacterial methanotrophs oxidize methane to methanol using the methane monooxygenase enzyme (MMO), which is further metabolized into formaldehyde using a methanol dehydrogenase (MDH). The main distinction between Type I or Type II methanotrophs is the pathway used for assimilation of formaldehyde into biomass; the ribulose monophosphate pathway for Type I methanotrophs, and the serine pathway for Type II methanotrophs.

[0107] Verrucomicrobial methanotrophs, however, oxidise methane terminally to carbon dioxide, which they then assimilate using the Calvin-Benson-Bassham cycle (Hou et aL, 2008, 'Complete genome sequence of the extremely acidophilic methanotroph isolate V4, Methylacidiphilum infernorum, a representative of the bacterial phylum Verrucomicrobia', Biology Direct, 3, 26, doi:10.1186 / 1745-6150-3-26; Khadem et al., 2011, 'Autotrophic methanotrophy in verrucomicrobia: Methylacidiphilum fumariolicum SolV uses the Calvin- Benson-Bassham cycle for carbon dioxide fixation', Journal of Bacteriology, 793(17), 4438- 4446, doi:10.1128 / JB.00407-11).

[0108] A culture of Methylacidiphilum sp. RTK17.1 was deposited at the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181, Ipsin-gil, Jeongeup-si, Jeolllabuk-do 56212, Republic of Korea on 19 February 2024, and was given accession number KCTC 15819BP. This is a recognised International Depositary Authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The depositor is TAUHARA NORTH NO. 2 TRUST, of Ngahere House, 283 Vaughan Road, Rotorua 3010, New Zealand.

[0109] The terms Methylacidiphilum sp. strain RTK17.1, Methylacidiphilum sp. RTK17.1, Methylacidiphilum RTK17.1, and RTK17.1 are used interchangeably herein. Methylacidiphilum sp. RTK17.1 is a verrucomicrobial methanotroph that was isolated in the Rotokawa region in New Zealand. It grows optimally at pH 2.5, 50°C (Tmax 60°C), oxidises CH4, fixes CO2, fixes N2 under nitrogen limiting conditions, accumulates glycogen, and rapidly consumes H2 under microaerophilic conditions. CO2 fixation occurs in cultures supplied with H2 as the sole reductant and O2 as the sole oxidant.

[0110] In some embodiments, the one or more strains of methanotrophic bacteria comprise, or consist of, a strain from the phylum Verrucomicrobiota, preferably from the order Methylacidiphilales, more preferably from the family Methylacidiphilaceae, more preferably from the genus Methylacidiphilum, Methylacidimicrobium, or Methylacidithermus, more preferably from the genus Methylacidiphilum, most preferably from the strain Methylacidiphilum sp. RTK17.1 KCTC accession number KCTC 15819BP dated 19 February 2024, or a derivative thereof.

[0111] In some embodiments, the one or more strains of methanotrophic bacteria have a 16S rRNA gene sequence that comprises a sequence with at least about 70% sequence identity to SEQ ID No: 1, such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID No: 1.

[0112] In some embodiments, the one or more strains of methanotrophic bacteria are aerobic. In some embodiments, the one or more strains of methanotrophic bacteria are thermophilic. In some embodiments, the one or more strains of methanotrophic bacteria are acidophilic. In some embodiments, the one or more strains of methanotrophic bacteria are thermoacidophilic.

[0113] In some embodiments, the one or more strains of acidophilic or thermoacidophilic methanotrophic bacteria are able to grow at, or have optimal growth at, a pH of less than about 5.0, such as less than about 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or less than about 0.0, and useful ranges may be selected between any of these values (for example, from about 0.0 to about 5.0, from about 0.0 to about 4.5, from about 0.0 to about 4.0, from about 0.0 to about 3.5, from about 0.0 to about 3.0, from about 0.0 to about 2.5, from about 0.5 to about 4.5, from about 0.5 to about 4.0, from about 0.5 to about 3.5, from about 0.5 to about 3.0, from about 0.5 to about 2.5, from about 1.0 to about 4.5, from about 1.0 to about 4.0, from about 1.0 to about 3.5, from about 1.0 to about 3.0, from about 1.0 to about 2.5, from about 1.8 to about 4.5, from about 1.8 to about 4.0, from about 1.8 to about 3.5, from about 1.8 to about 3.0, or from about 1.8 to about 2.5)).

[0114] In some embodiments, the one or more strains of thermophilic or thermoacidophilic methanotrophic bacteria are able to grow at, or have optimal growth at, a temperature of at least about 35°C, such as at least about 40°C, at least about 42°C, at least about 44°C, at least about 46°C, at least about 48°C, at least about 50°C, at least about 52°C, at least about 54°C, at least about 56°C, at least about 58°C, at least about 60°C, at least about 62°C, at least about 64°C, at least about 66°C, at least about 68°C, at least about 70°C, at least about 72°C, at least about 74°C, at least about 76°C, at least about 78°C, or at least about 80°C, and useful ranges may be selected between any of these values (for example, from about 40°C to about 80°C, from about 40°C to about 70°C, from about 40°C to about 60°C, from about 40°C to about 58°C, from about 40°C to about 56°C, from about 40°C to about 54°C, from about 40°C to about 52°C, from about 40°C to about 50°C, from about 46°C to about 80°C, from about 46°C to about 70°C, from about 46°C to about 60°C, from about46°C to about 58°C, from about 46°C to about 56°C, from about 46°C to about 54°C, from about 46°C to about 52°C, from about 46°C to about 50°C, from about 50°C to about 66°C, from about 50°C to about 64°C, from about 50°C to about 62°C, from about 50°C to about 60°C, from about 50°C to about 58°C, from about 50°C to about 56°C, or from about 50°C to about 54°C).

[0115] In some embodiments, the one or more strains of methanotrophic bacteria are capable of fixing carbon dioxide. In some embodiments, the one or more strains of methanotrophic bacteria are capable of fixing nitrogen under nitrogen limiting conditions. In some embodiments, the one or more strains of methanotrophic bacteria are capable of consuming hydrogen under microaerophilic conditions.3. Phototrophs

[0116] Phototrophs are organisms that are capable of converting light into energy. Preferred phototrophs are photoautotrophs that can perform photosynthesis including (but are not limited to) algae such as microalgae. One exemplary embodiment of a phototroph is microalgae from the genus Galdieria. Accordingly, in some embodiments, the one or more strains of phototroph comprises, or consists of, a strain of algae, preferably a microalgae, more preferably from the class Cyanidiophyceae, more preferably a species of Galdieria, or most preferably Galdieria sp. RTK37.1 KCTC accession number KCTC 15846BP dated 7 March 2024, or a derivative thereof.

[0117] Galdieria spp. are unicellular thermoacidophilic red algae belonging to the class Cyanidiophyceae, which can typically grow autotrophically, heterotrophically, and mixotrophically (Schmidt et al., 2005, 'Heterotrophic high cell-density fed-batch cultures of the phycocyanin-producing red alga Galdieria sulphuraria', Biotechnology and Bioengineering, 90(1), 77-84.). They favour growth in cryptoendolithic habitats, which are usually challenging environments for photosynthetic organisms due to restricted access to light. They are typically tolerant of high concentrations of salt and heavy metals (Lopez et al., 2019, 'Production of Polyunsaturated Fatty Acids and Lipids from Autotrophic, Mixotrophic and Heterotrophic cultivation of Galdieria sp. strain USBA-GBX-832.' Scientific Reports, 9(1), 10791. doi:10.1038 / s41598-019-46645-3).

[0118] Galdieria spp. are often found in hostile environments like volcanic hot sulphur springs and solfatara soils. They can grow in temperatures up to 56°C, and pH as acidic as 0.0 (Marquardt & Rhiel, 1997, 'The membrane-intrinsic light-harvesting complex of the red alga Galdieria sulphuraria (formerly Cyanidium caldarium): biochemical and immunochemical characterization', Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1320(2), 153-164. doi:https: / / doi.org / 10.1016 / 50005-2728(97)00020-0; Vitova et al., 2016, 'Lipidomic analysis ofthe extremophilic red alga Galdieria sulphuraria in response to changes in pH.' Algal Research, 13, 218-226. doi:10.1016 / j.algal.2O15.12.005).

[0119] A distinguishing characteristic of Galdieria spp. is their capacity to grow heterotrophically on a variety of reduced carbon substrates, including disaccharides, hexoses, pentoses, amino acids, TCA cycle intermediates, and some organic acids (Gross & Schnarrenberger, 1995, 'Heterotrophic growth of two strains of the acido-thermophilic red alga Galdieria sulphuraria.' Plant and Cell Physiology, 36(4), 633-638; Vitova et al., 2016, 'Lipidomic analysis of the extremophilic red alga Galdieria sulphuraria in response to changes in pH.' Algal Research, 13, 218-226. doi:10.1016 / j.algal.2015.12.005).

[0120] A culture of Galdieria sp. RTK37.1 was deposited at the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181, I psin-gil, Jeongeup-si, Jeolllabuk-do 56212, Republic of Korea on 7 March 2024, and was given accession number KCTC 15846BP. This is a recognised International Depositary Authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The depositor is TAUHARA NORTH NO. 2 TRUST, of Ngahere House, 283 Vaughan Road, Rotorua 3010, New Zealand.

[0121] The terms Galdieria sp. strain RTK37.1, Galdieria sp. RTK37.1, Galdieria RTK37.1, and RTK37.1 are used interchangeably herein. Galdieria sp. RTK37.1 is an extremophilic unicellular red alga with moderate thermophily that is tolerant of 100% CO2 in the headspace, and grows optimally in acidic pH. It is capable of growing photoautotrophically, chemoheterotrophically, or mixotrophically.

[0122] In some embodiments, the one or more strains of phototroph comprise, or consist of, a strain from the class Cyanidiophyceae, preferably from the family Cyanidiaceae or Galdieriaceae, more preferably from the genera Cyanidioschyzon, Cyanidium, Galdieria or Cyanidiococcus, more preferably from the genus Galdieria, most preferably Galdieria sp. RTK37.1 KCTC accession number KCTC 15846BP dated 7 March 2024, or a derivative thereof.

[0123] In some embodiments, the one or more strains of phototroph have an 18S rRNA gene sequence that comprises a sequence with at least about 60% sequence identity to SEQ ID No: 2, such as at least about 75%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID No: 2.

[0124] In some embodiments, the one or more strains of phototroph are thermophilic. In some embodiments, the one or more strains of phototroph are acidophilic. In some embodiments, the one or more strains of phototroph are thermoacidophilic.

[0125] In some embodiments, the one or more strains of acidophilic or thermoacidophilic phototroph are able to grow at, or have optimal growth at, a pH of less than about 5.0, such as less than about 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 or less than about 0.0, and useful ranges may be selected between any of these values (for example, from about 0.0 to about 5.0, from about 0.0 to about 4.5, from about 0.0 to about 4.0, from about 0.0 to about 3.5, from about 0.0 to about 3.0, from about 0.0 to about 2.5, from about 0.5 to about 4.5, from about 0.5 to about 4.0, from about 0.5 to about 3.5, from about 0.5 to about 3.0, from about 0.5 to about 2.5, from about 1.0 to about 4.5, from about 1.0 to about 4.0, from about 1.0 to about 3.5, from about 1.0 to about 3.0, from about 1.0 to about 2.5, from about 1.8 to about 4.5, from about 1.8 to about 4.0, from about 1.8 to about 3.5, from about 1.8 to about 3.0, or from about 1.8 to about 2.5).

[0126] In some embodiments, the one or more strains of thermophilic or thermoacidophilic phototroph are able to grow at, or have optimal growth at, a temperature of at least about 35°C, such as at least about 40°C, at least about 42°C, at least about 44°C, at least about 46°C, at least about 48°C, at least about 50°C, at least about 52°C, at least about 54°C, at least about 56°C, at least about 58°C, or at least about 60°C, and useful ranges may be selected between any of these values (for example, from about 40°C to about 60°C, from about 40°C to about 58°C, from about 40°C to about 56°C, from about 40°C to about 54°C, from about 40°C to about 52°C, from about 40°C to about 50°C, from about 42°C to about 60°C, from about 42°C to about 58°C, from about 42°C to about 56°C, from about 42°C to about 54°C, from about 42°C to about 52°C, from about 42°C to about 50°C, from about 44°C to about 60°C, from about 44°C to about 58°C, from about 44°C to about 56°C, from about 44°C to about 54°C, from about 44°C to about 52°C, from about 44°C to about 50°C, from about 46°C to about 60°C, from about 46°C to about 58°C, from about 46°C to about 56°C, from about 46°C to about 54°C, from about 46°C to about 52°C, or from about 46°C to about 50°C).4. Culture conditions

[0127] Culturing and / or coculturing will typically take place in a culture medium, such as an aqueous culture medium. Accordingly, in some embodiments the culturing and / or coculturing occurs in a culture medium, preferably an aqueous culture medium.

[0128] A wide variety of culture mediums are available, and the skilled person will be able to select a medium according to the requirements of the particular methanotrophic bacteria and / or phototroph used. One exemplary culture medium suitable for culturing and / or coculturing Methylacidiphilum sp. RTK17.1 and / or Galdieria sp. RTK37.1 is modified V4 medium, as described in Example 1.

[0129] Culture medium is typically aqueous (i.e. comprises water) and usually contains a nitrogen source and various other components to support growth of the methanotrophic bacteria and / or phototroph. For example, culture medium will typically comprise sources of phosphate, magnesium, iron, and may comprise other trace metals and / or trace elements. Culture medium may also comprise a carbon source, however as the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph may be able to fix carbon from carbon dioxide, an additional carbon source may not be required.

[0130] A variety of nitrogen sources are known in the art, and the skilled person will be able to select one appropriate to the methanotrophic bacteria and / or the phototroph used. In some embodiments, the methanotrophic bacteria may be capable of fixing nitrogen gas, and therefore in these embodiments, nitrogen gas (N2) may be used as a nitrogen source. In some embodiments, the culture medium may contain ammonium and / or nitrate as a nitrogen source. Preferably, the nitrogen source comprises ammonium. Chemical compounds that produce ammonium and / or nitrate ions when dissolved in water are readily available.

[0131] It will be appreciated that components of the culture medium (for example, nitrogen, trace metals, and / or trace elements) may become depleted over time, particularly as biomass is harvested. Accordingly, it may be necessary to replenish these components of the culture medium from time to time. Similarly, the pH of the culture medium may change over time (for example, becoming more acidic), so acids, bases, and / or pH buffers may be added to adjust or maintain the pH.

[0132] It will be appreciated that different strains of methanotrophic bacteria and phototrophs may have different culture requirements. For example, the optimal culture medium composition, culture temperature, pH, light intensity, dissolved oxygen concentration, agitation rate, and so on, may differ depending on the specific strain(s) used. The skilled person will be able to determine and / or select suitable parameters based on the particular strain(s) of methanotrophic bacteria and phototroph used.

[0133] In some embodiments, the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph are thermophilic or thermoacidophilic. Accordingly, in some embodiments, culturing the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph comprises culturing at a temperatureof at least about 35°C, such as at least about 40°C, at least about 42°C, at least about 44°C, at least about 46°C, at least about 48°C, at least about 50°C, at least about 52°C, at least about 54°C, at least about 56°C, at least about 58°C, or at least about 60°C, and useful ranges may be selected between any of these values (for example, from about 35°C to about 60°C, from about 40°C to about 60°C, from about 40°C to about 56°C, from about 40°C to about 52°C, from about 40°C to about 50°C, from about 42°C to about 60°C, from about 42°C to about 56°C, from about 42°C to about 52°C, from about 42°C to about 50°C, from about 44°C to about 60°C, from about 44°C to about 56°C, from about 44°C to about 52°C, or from about 44°C to about 50°C).

[0134] In some embodiments, the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph are acidophilic or thermoacidophilic. Accordingly, in some embodiments the culture medium has a pH of less than about 6.0, such as less than about 5.5, less than about 5.0, less than about 4.5, less than about 4.0, less than about 3.5, less than about 3.0, less than about 2.5, less than about 2.5, less than about 2.3, less than about 2.2, less than about 2.1, less than about 2.0, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.0, or less than about 0.5, or less than about 0.0, and useful ranges may be selected between any of these values (for example, from about 0.5 to about 6, from about 0.5 to about 5.5, from about 0.5 to about 5, from about 0.5 to about 4.5, from about 0.5 to about 4, from about 0.5 to about 3.5, from about 0.5 to about 3, from about 0.5 to about 2.5, from about 1 to about 6, from about 1 to about 5.5, from about 1 to about 5, from about 1 to about 4.5, from about 1 to about 4, from about 1 to about 3.5, from about 1 to about 3, from about 1 to about 2.5, from about 1.5 to about 6, from about 1.5 to about 5.5, from about 1.5 to about 5, from about 1.5 to about 4.5, from about 1.5 to about 4, from about 1.5 to about 3.5, from about 1.5 to about 3, from about 1.5 to about 2.5, from about 1.8 to about 6, from about 1.8 to about 5.5, from about 1.8 to about 5, from about 1.8 to about 4.5, from about 1.8 to about 4, from about 1.8 to about 3.5, from about 1.8 to about 3, from about 1.8 to about 2.5, from about 2 to about 6, from about 2 to about 5.5, from about 2 to about 5, from about 2 to about 4.5, from about 2 to about 4, from about 2 to about 3.5, from about 2 to about 3, or from about 2 to about 2.5). Preferably, the culture medium has a pH of about 2.5.

[0135] In a preferred embodiment, the one or more strains of methanotrophic bacteria and the one or more strains of phototroph are thermoacidophilic. Thermoacidophilic microorganisms are particularly preferred for the methods and / or systems of the invention, as they may be cultured and / or cocultured at higher temperatures and lower pH than other microorganisms. This can result in a decreased need for cooling of the input gas, and also provides resistance to contamination by unwanted microorganisms, as most such contaminating microorganisms are unable to grow, or have reduced growth rate, at thesetemperatures and / or pHs. Reduced risk of contamination by unwanted microorganisms may be particularly important in an industrial context, in which aseptic conditions may be difficult and / or expensive to maintain.

[0136] Culturing and / or coculturing at temperatures above about 45°C and at a pH of less than about 4.0 (preferably less than about 3.0) is particularly preferred, as it is believed that such conditions may provide increased resistance to contamination by unwanted microorganisms than culturing and / or coculturing at lower temperatures and higher pHs.

[0137] As the phototrophs are able to convert light into chemical energy, culturing and / or coculturing the phototrophs should take place in the presence of light. Various sources of light are known in the art, and include artificial light sources (for example, incandescent lights, fluorescent lights, vapour lamps, and light-emitting diodes (LEDs)) and natural light sources (for example, sunlight). Sunlight may be used as a light source in the methods and systems of the invention by using a material that is permeable to light, preferably permeable to the wavelengths of light involved in photosynthesis. These wavelengths include light falling within the blue (425-450 nm) and red (600-700 nm) ranges. Glass and various plastics, such as acrylics (polymethlamethacrylate), butyrate (cellulose acetate butyrate), lexan (polycarbonate), PETG (glycol modified polyethylene terphthalate), and combinations thereof are impermeable to fluids and are permeable to visible light, making them suitable housing materials.

[0138] In some embodiments the one or more strains of phototroph are exposed to sunlight. In some embodiments, the one or more strains of phototroph are exposed to light from an artificial light source.

[0139] The skilled person will be able to select an appropriate intensity and wavelength of light, depending on the requirements of the particular strain(s) of phototroph and the system / apparatus used. Light intensity may be measured using means known in the art, for example using a LI-250A Light Meter (LI-COR Biosciences). Preferably, light intensity is measured at the centre of the vessel or chamber in which the phototroph is to be cultured.

[0140] In some embodiments, the light has an intensity of at least about 30 pmol photons m’2s’1, such as at least about 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 450, 500, 550, or at least about 600 pmol photons m-2s’1, and useful ranges may be selected between any of these values (for example, from about 30 to about 600, from about 30 to about 500, from about 30 to about 400, from about 30 to about 300, from about 60 to about 600, from about 60 to about 500, from about 60 to about 400, or from about 60 to about 300 pmol photons m’2s’1).

[0141] The one or more strains of methanotrophic bacteria require oxygen for optimal growth and methane oxidation. Furthermore, the inventors have surprisingly found that the phototrophs may require a minimum oxygen concentration to avoid coproporphyrin production and / or pigment loss. Accordingly, in some embodiments, the culture medium comprises at least about 0.2 mg / L of dissolved oxygen, such as at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or at least about 2.0 mg / L of dissolved oxygen, and useful ranges may be selected between any of these values (for example, from about 0.2 to about 2 mg / L, from about 0.2 to about 1.5 mg / L, from about 0.2 to about 1 mg / L, from about 0.4 to about 2 mg / L, from about 0.4 to about 1.5 mg / L, from about 0.4 to about 1 mg / L, from about 0.6 to about 2 mg / L, from about 0.6 to about 1.5 mg / L, from about 0.6 to about 1 mg / L, from about 0.8 to about 2 mg / L, from about 0.8 to about 1.5 mg / L, or from about 0.8 to about 1 mg / L). Dissolved oxygen concentration may be adjusted by various means known in the art, for example by altering agitation rate, oxygen concentration of a headspace gas (for example, the input gas and / or the intermediate gas), pressure, and / or gas flow rate.

[0142] The culturing and / or coculturing may take place at ambient pressure, or it may take place at increased pressure. This may be achieved, for example, by pressurising a bioreactor with the input or intermediate gas. In some embodiments, the culturing and / or coculturing takes place at a pressure above atmospheric pressure. In some embodiments, the culturing and / or coculturing takes place at a pressure of at least about 1 mbar above atmospheric pressure, such as at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or at least about 5000 mbar above atmospheric pressure, and useful ranges may be selected between any of these values (for example, from about 1 to about 5000, from about 1 to about 4000, from about 1 to about 3000, from about 1 to about 2000, from about 1 to about 1500, from about 1 to about 1200, from about 50 about 4000, from about 50 about 3000, from about 50 about 2000, from about 50 about 1500, from about 50 about 1200, from about 100 about 4000, from about 100 about 3000, from about 100 about 2000, from about 100 about 1500, from about 100 about 1200, from about 500 about 4000, from about 500 about 3000, from about 500 about 2000, from about 500 about 1500, or from about 500 about 1200 mbar above atmospheric pressure).

[0143] The methanotrophic bacteria and the phototroph may be present in various different ratios when initially setting up the culture(s) or coculture(s) and / or during use. It will be appreciated that the ratio used to initiate a coculture may differ from the steady-state ratio present once a stable coculture has been established. It is anticipated that microorganisms will naturally equilibrate to a particular steady-state ratio, depending on theculture conditions and other parameters used. A given steady-state ratio may therefore be achieved by varying the culture conditions and other parameters.

[0144] Mass ratios of methanotrophic bacteria and phototroph may preferably be calculated in terms of grams dry weight of methanotrophic bacteria to grams dry weight of phototroph (gDw:gow).

[0145] Mass of methanotrophic bacteria and / or phototroph may be measured gravimetrically using techniques known in the art. One exemplary method is provided herein.

[0146] 15 mL aliquots of liquid culture containing the methanotrophic bacteria and / or phototroph are provided. These are vacuum-filtered on pre-weighted 0.22 pm nylon membrane filters (MicroScience, Australia). The filters are then washed 3 times by vacuumfiltering 10 mL of deionized water. The filters are dried for 12 hours at 95°C, weighed, and the dry weight mass calculated by difference.

[0147] Alternatively, mass of methanotrophic bacteria and / or phototroph may be estimated by measuring the volume and optical density at 600 nm (ODeoo) of a liquid culture, and using a conversion factor. Conversion factors can be derived by measuring the ODeoo of cultures at various concentrations and determining the dry weight mass gravimetrically as described above. The authors have found that one unit of ODeoo is equivalent to 0.308 gow L’1for Galdieria sp. RTK37.1, and 0.436 gow L’1for Methylacidiphilum sp. RTK17.1.

[0148] Unexpectedly, the inventors have found that higher initial mass ratios of methanotrophic bacteria to thermophilic phototroph, when in batch culture, are detrimental to the growth of the phototroph, causing pigment loss, coproporphyrin production, and reduced or ceased growth of the phototroph. This ultimately leads to a steep decline in biomass production and net carbon fixation. Without wishing to be bound by theory, it is believed that the methanotrophic bacteria have a higher affinity for oxygen than the phototroph, and at higher mass ratios are therefore able to monopolise the dissolved oxygen. It is also believed that this effect can be avoided by ensuring oxygen concentrations are sufficiently high.

[0149] The inventors have found that initial dry weight mass ratios of methanotrophic bacteria to phototroph that are less than 0.5 are preferable, and particularly mass ratios less than 0.23.

[0150] In some embodiments the dry weight mass ratio of methanotrophic bacteria to phototroph is less than about 0.50, such as less than about 0.45, less than about 0.40, less than about 0.35, less than about 0.30, less than about 0.25, less than about 0.24, less than about 023, less than about 0.22, less than about 0.21, less than about 0.20, less than about0.19, less than about 0.18, less than about 0.17, less than about 0.16, less than about 0.15, less than about 0.14, less than about 0.13, less than about 0.12, less than about 0.11, or less than about 0.10, and useful ranges may be selected between any of these values (for example, from about 0.10 to about 0.50, from about 0.10 to about 0.40, from about 0.10 to about 0.30, from about 0.10 to about 0.25, from about 0.10 to about 0.24, from about 0.10 to about 0.22, from about 0.10 to about 0.20, from about 0.10 to about 0.18, from about 0.14 to about 0.50, from about 0.14 to about 0.40, from about 0.14 to about 0.30, from about 0.14 to about 0.25, from about 0.14 to about 0.24, from about 0.14 to about 0.22, from about 0.14 to about 0.20, from about 0.14 to about 0.18, from about 0.16 to about 0.50, from about 0.16 to about 0.40, from about 0.16 to about 0.30, from about 0.16 to about 0.25, from about 0.16 to about 0.24, from about 0.16 to about 0.22, from about 0.16 to about 0.20, or from about 0.16 to about 0.18). In some embodiments, this ratio is the initial dry weight mass ratio when establishing a coculture. In other embodiments, this ratio is the steady-state dry weight mass ratio.

[0151] In some embodiments, the dry weight mass ratio of methanotrophic bacteria to phototroph is from about 1000000 to about 0.000001, for example from about 1000000 to about 0.00001, from about 1000000 to about 0.0001, from about 1000000 to about 0.001, from about 1000000 to about 0.01, from about 1000000 to about 0.1, from about 1000000 to about 1, from about 100000 to about 0.000001, from about 10000 to about 0.000001, from about 1000 to about 0.000001, from about 100 to about 0.000001, from about 10 to about 0.000001, from about 1 to about 0.000001, from about 1000 to about 0.001, from about 1000 to about 0.01, from about 1000 to about 0.1, from about 1000 to about 1, from about 100 to about 0.001, from about 100 to about 0.01, from about 100 to about 0.1, from about 100 to about 1, from about 10 to about 0.001, from about 10 to about 0.01, from about 10 to about 0.1, from about 10 to about 1, from about 1 to about 0.001, from about 1 to about 0.01, or from about 1 to about 0.1 . In some embodiments, this ratio is the steady-state dry weight mass ratio.5. Input gas

[0152] In some embodiments, the input gas comprises one or more of the following: methane, carbon monoxide, carbon dioxide, oxygen, hydrogen, nitrogen, hydrogen sulfide, and / or air.

[0153] In some embodiments, the input gas comprises methane. In some embodiments, the input gas comprises at least about 0.1% v / v methane, such as at least about 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or at least about 20% v / v methane, anduseful ranges may be selected between any of these values (for example, from about 0.1% to about 20%, from about 0.1% to about 15%, from about 0.1% to about 10%, from about 0.1% to about 5%, from about 0.1% to about 4%, from about 0.1% to about 3%, from about 0.1% to about 2%, from about 0.1% to about 1%, from about 0.5% to about 20%, from about 0.5% to about 15%, from about 0.5% to about 10%, from about 0.5% to about 5%, from about 0.5% to about 4%, from about 0.5% to about 3%, from about 0.5% to about 2%, from about 0.5% to about 1%, from about 1.0% to about 20%, from about 1.0% to about 15%, from about 1.0% to about 10%, from about 1.0% to about 5%, from about 1.0% to about 4%, from about 1.0% to about 3%, from about 1.0% to about 2%, or from about 1.0% to about 1.5% v / v).

[0154] The methane may come from a variety of sources including, but not limited to: methane from oil or natural gas processing; methane from coal mining; geologically or geothermally produced or emitted methane; methane from biomass burning; methane from biological processes such as landfill or wastewater treatment; or methane from manure such as livestock manure.

[0155] In some embodiments, the input gas comprises oxygen. In some embodiments, the input gas comprises at least about 1.0% v / v oxygen, such as at least about 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or at least about 20% v / v oxygen, and useful ranges may be selected between any of these values (for example, from about 1.0% to about 20%, from about 1.0% to about 15%, from about 1.0% to about 10%, from about 1.0% to about 5%, from about 1.0% to about 4%, from about 1.0% to about 3%, from about 1.0% to about 2.5%, from about 1.0% to about 2.2%, from about 1.5% to about 20%, from about 1.5% to about 15%, from about 1.5% to about 10%, from about 1.5% to about 5%, from about 1.5% to about 4%, from about 1.5% to about 3%, from about 1.5% to about 2.5%, or from about 1.5% to about 2.2%). Preferably, the input gas comprises about 3% v / v oxygen.

[0156] In some embodiments, the input gas comprises carbon dioxide. In some embodiments, the input gas comprises at least about 10% v / v carbon dioxide, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or at least about 98% v / v carbon dioxide, and useful ranges may be selected between any of these values (for example, from about 10% to about 98%, from about 10% to about 94%, from about 10% to about 90%, from about 10% to about 86%, from about 10% to about 80%, from about 10% to about 75%, from about 10% to about 70%, from about 10% to about 65%, from about 10% to about 60%, from about 20% to about 98%, from about 20% to about 94%, from about 20% to about 90%, from about20% to about 86%, from about 20% to about 80%, from about 20% to about 75%, from about 20% to about 70%, from about 20% to about 65%, from about 20% to about 60%, from about 30% to about 98%, from about 30% to about 94%, from about 30% to about 90%, from about 30% to about 86%, from about 30% to about 80%, from about 30% to about 75%, from about 30% to about 70%, from about 30% to about 65%, from about 30% to about 60%, from about 40% to about 98%, from about 40% to about 94%, from about 40% to about 90%, from about 40% to about 86%, from about 40% to about 80%, from about 40% to about 75%, from about 40% to about 70%, from about 40% to about 65%, from about 40% to about 60%, from about 50% to about 98%, from about 50% to about 94%, from about 50% to about 90%, from about 50% to about 86%, from about 50% to about 80%, from about 50% to about 75%, from about 50% to about 70%, from about 50% to about 65%, or from about 50% to about 60% v / v). Preferably, the input gas comprises about 73% v / v carbon dioxide.

[0157] The carbon dioxide may come from sources including, but not limited to: carbon dioxide from the gasification of organic matter; carbon dioxide from the calcination of limestone, CaCOs, to produce quicklime, CaO; carbon dioxide from methane steam reforming, such as the carbon dioxide by-product from ammonia, methanol, or hydrogen production; carbon dioxide from combustion, incineration, or flaring; carbon dioxide byproduct of anaerobic or aerobic fermentation of sugar; carbon dioxide by-product of a methanotrophic bioprocess; carbon dioxide from waste water treatment; carbon dioxide byproduct from sodium phosphate production; geologically or geothermally produced or emitted carbon dioxide; carbon dioxide removed from acid gas or natural gas.

[0158] In some embodiments, the carbon dioxide has been removed from an industrial flue gas, produced by a chemical process such as steel making or cement production, or intercepted from a geological source that would otherwise naturally emit into the atmosphere. In some embodiments, the carbon dioxide is carbon dioxide and / or bicarbonate and / or carbonate dissolved in sea water or other bodies of surface or underground water. In some such embodiments, inorganic carbon may be introduced dissolved in liquid water and / or as a solid. In some embodiments, the carbon dioxide is captured from the atmosphere. In some embodiments, the carbon dioxide is emitted by a geological feature such as, but not limited to, geothermal and / or hydrothermal vents.

[0159] Gases that comprises a mixture of flammable gas(es) and oxygen may form a flammable or even explosive mixture. For example, input gas comprising methane and oxygen may form an explosive mixture, if the concentrations of methane and oxygen are sufficiently high. Methane-oxygen mixtures become explosive between 5% and 15% methane by volume in air. Janes et al., provides some discussion of the flammability of such mixtures,and is hereby incorporated by reference in its entirety (Janes et al., 2011, 'Experimental study of CH4 / O2 / CO2 mixtures flammability.' 1 lAIChE - 2011 AIChE Spring Meeting and 7th Global Congress on Process Safety, Conference Proceedings).

[0160] Explosive mixtures may be avoided by using a sufficiently low concentration of methane and / or oxygen. Preferably, the input gas is not an explosive mixture. Without wishing to be bound by theory, it is believed that an explosive mixture can be avoided by the use of the methods and systems of the invention. Specifically, it is believed that the phototroph is able to produce oxygen in situ by means of photosynthesis, thereby providing supplemental oxygen that enables further growth of the methanotrophic bacteria, without requiring a high concentration of oxygen in the input gas. Additionally, is some embodiments the methanotrophic bacteria have a high affinity for oxygen, which prevents the accumulation of O2 gas when methane is present.

[0161] The applicant anticipates that a wide variety of sources of input gas may be useful in the methods and systems of the invention. An input gas may comprise gas from a single source, or it may comprise gas from a combination of any two or more sources.

[0162] In some embodiments, the input gas comprises a gas from a carbon capture and storage (CCS) system. In some embodiments, the input gas comprises a gas (such as CO2 and / or O2) that is captured from the atmosphere.

[0163] In some embodiments, the input gas comprises a waste gas from an industrial process, for example an industrial flue gas. In some embodiments, the industrial process is power generation such as geothermal power generation, oil production, natural gas production, biogas production, or smelting.

[0164] In some embodiments, the input gas comprises gas produced by the microbial degradation of organic materials, for example during wastewater or sewage treatment, from landfill, or from composting. An exemplary wastewater is liquid effluent from an anaerobic digester, such as an anaerobic sewage or sludge digester, and an exemplary wastewater source is the liquid reservoir of an anaerobic digester, such as an anaerobic sewage or sludge digester. Particular examples of wastewater include animal farm wastewater, vegetable farm wastewater, food processing plant wastewater, winery wastewater, landfill wastewater, and fishery wastewater.

[0165] In some embodiments, an input gas is used in the methods and / or systems of the invention directly. In other embodiments, one or more additional gases are added to a gas source to provide an input gas. The one or more additional gases may be from any suitable source, including (but not limited to) commercially available gases, the gas sourcesdescribed herein, and / or air. In some embodiments, the one or more additional gases may comprise a recirculated gas from the methods and / or systems of the invention. The composition of the input gas may be thereby adjusted to suit the requirements of the methanotrophic bacteria to phototroph, for example by adjusting the oxygen, methane, nitrogen and / or carbon dioxide content.

[0166] Waste gas streams from industrial processes may be produced at a higher than ambient temperature. Such waste gas streams may previously have required cooling so as to avoid overheating microbial cultures. However, without wishing to be bound by theory, it is believed that the use of the methanotrophic bacteria and phototrophs of the invention may allow an input gas to be used directly from an industrial process, without needing cooling, or at least with a reduced need for cooling.

[0167] Accordingly, in some embodiments the input gas is at a higher than ambient temperature. For example, in some embodiments the input gas is at a temperature of at least about 35°C, such as at least about 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or at least about 100°C, and useful ranges may be selected between any of these values (for example, from about 35°C to about 100°C, from about 35°C to about 90°C, from about 35°C to about 80°C, from about 35°C to about 70°C, from about 40°C to about 100°C, from about 40°C to about 90°C, from about 40°C to about 80°C, from about 40°C to about 70°C, from about 45°C to about 100°C, from about 45°C to about 90°C, from about 45°C to about 80°C, from about 45°C to about 70°C, from about 50°C to about 100°C, from about 50°C to about 90°C, from about 50°C to about 80°C, from about 50°C to about 70°C, from about 55°C to about 100°C, from about 55°C to about 90°C, from about 55°C to about 80°C, from about 55°C to about 70°C, from about 60°C to about 100°C, from about 60°C to about 90°C, from about 60°C to about 80°C, from about 60°C to about 70°C).

[0168] Waste gas streams from industrial processes may contain gases such as carbon dioxide, H2S, NOX, and SOXthat can cause difficulties when culturing microorganisms. These gases may acidify liquid culture medium to an extent that inhibits the growth and diminishes the productivity of many microorganisms. However, without wishing to be bound by theory, it is believed that the methanotrophic bacteria and phototrophs described herein may be better able to tolerate acidic pH and / or the presence of these gases.

[0169] In some embodiments, the input gas comprises carbon dioxide, carbon monoxide, NOX, SOX, H2S, or any combination of any two or more of these. It is believed that, in certain embodiments of the invention, the methanotrophic bacteria and phototrophs may be better able to tolerate the presence of such gases, resulting in a reduced need for pretreatment to remove or reduce such gases.

[0170] For example, the inventors have found that Methylacidiphilum sp. RTK17.1 is able to grow in the presence of at least about 600 ppm H2S.

[0171] Accordingly, in some embodiments the input gas comprises at least about 100 ppm H2S, such as at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or at least about 6000 ppm H2S, and useful ranges may be selected between any of these values (for example, from about 100 to about 6000, from about 100 to about 5000, from about 100 to about 4000, from about 100 to about 3000, from about 100 to about 2000, from about 100 to about 1000, from about 100 to about 800, from about 100 to about 600, from about 300 to about 6000, from about 300 to about 5000, from about 300 to about 4000, from about 300 to about 3000, from about 300 to about 2000, from about 300 to about 1000, from about 300 to about 800, or from about 300 to about 600).

[0172] In some embodiments, an input gas is used directly from an industrial process without pre-treatment to remove or reduce carbon dioxide, carbon monoxide, NOX, SOX, and / or H2S. contaminants. In some embodiments, the input gas has not been subjected to pre-treatment to remove or reduce the amount of carbon dioxide, carbon monoxide, NOX, SOX, and / or H2S. In some embodiments, the input gas is an exhaust gas from an industrial process that is used directly in the methods and / or systems of the invention.6. Output gas

[0173] The inventors have found that culturing and / or coculturing one or more strains of methanotrophic bacteria and one or more strains of phototroph allows the microorganisms to grow using gases such as methane and carbon dioxide as an energy and carbon source. This allows the methane and / or carbon dioxide to be converted into biomass, while also decreasing the methane and / or carbon dioxide content of the output gas.

[0174] Accordingly, in some embodiments, the output gas has a reduced methane content compared to the input gas. In some embodiments, the output gas has a methane content that is less than about 50% of the methane content of the input gas, such as less than about 40%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% of the methane content of the input gas.

[0175] In some embodiments, the output gas contains less than about 10% v / v methane, such as less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.10%, 0.05%, or less than about 0.01% v / v methane. In some embodiments, the output gas contains substantially no methane.

[0176] In some embodiments, the output gas is primarily CO2. Such purified CC gas streams are useful in various applications known in the art. In some embodiments, the output gas contains at least about 50% v / v CO2, for example at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least about 99.9% v / v CO2, and useful ranges may be selected between any of these values (for example, from about 50% to about 99.9%, from about 50% to about 99%, from about 50% to about 95%, from about 50% to about 90%, from about 50% to about 85%, from about 50% to about 80%, from about 70% to about 99.9%, from about 70% to about 99%, from about 70% to about 95%, from about 70% to about 90%, from about 70% to about 85%, from about 70% to about 80%, from about 90% to about 99.9%, from about 90% to about 99%, from about 90% to about 95%, from about 95% to about 99.9%, or from about 95% to about 99%). In some embodiments, such output gas also comprises less than about 5%, 4%, 3%, 2%, 1.0%, 0.5%, 0.10%, 0.05%, or less than about 0.01% v / v methane.

[0177] In other embodiments, the output gas has a reduced CO2 content compared to the input gas. In some embodiments, the output gas has a CO2 content that is less than about 90% of the CO2 content of the input gas, such as less than about 80%, 70%, 60%, 50%, 40%, 30%, 25%, or less than about 20% of the CO2 content of the input gas.

[0178] In some embodiments, the output gas contains less than about 60% v / v CO2, such as less than about 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less than about 1% v / v CO2.

[0179] In some embodiments, the methods and / or systems of the invention remove at least about 1 kg / day of methane from the input gas, such as at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or at least about 1000 kg / day, and useful ranges may be selected between any of these values (for example, from about 1 to about 1000, from about 1 to about 500, from about 1 to about 100, from about 100 to about 1000, from about 100 to about 500, or from about 500 to about 1000 kg / day of methane).

[0180] In some embodiments, the methods and / or systems of the invention remove at least about 100 kg / day of CO2 from the input gas, such as at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or at least about 100000 kg / day, and useful ranges may be selected between any of these values (for example, from about 100 to about 100000, from about 100 to about 50000, from about 100 to about 10000, from about 100 to about 5000, from about 1000 to about 100000, from about 1000 to about 50000, from about 1000 to about 10000, from about 1000 to about 5000, from about 10000 to about 100000, from about 10000 to about 50000, or from about 50000 to about 100000 kg / day of CO2).7. Systems

[0181] The methods and / or systems of the invention comprise culturing and / or coculturing one or more strains of methanotrophic bacteria and one or more strains of phototroph. Culturing and / or coculturing may take place in any suitable apparatus, such as a bioreactor.

[0182] One exemplary embodiment of a system of the invention, which may be useful in a method of the invention, comprises a system adapted to receive an input gas and provide an output gas. The system may also be adapted to receive an input liquid (such as liquid culture medium or a component thereof), and / or to provide an output liquid (such as liquid culture medium, optionally comprising a biological component such as biomass). In some embodiments, the system comprises one or more bioreactors.

[0183] A bioreactor is an apparatus in which a biological reaction or process is carried out. Bioreactors are widely used for carrying out biological reactions or processes on an industrial scale. Bioreactors designed for the growth or fermentation of microorganisms are well known in the art, and are particularly suitable for the methods and systems of the invention. Some exemplary embodiments of bioreactors include the BioFlo 110 (New Brunswick Scientific), Biostat D-DCU STR reactor (Sartorius), and a tubular photobioreactor (Smith, 2020, "Optimisation of eicosapentaenoic acid productivity from a New Zealand microalga in a tubular photobioreactor", doctoral thesis, University of Canterbury, doi: 10.26021 / 10197). The size and configuration of the bioreactor may be selected according to need, for example depending on the volume and composition of the input gas to be processed and / or on the type and amount of biomass to be produced.

[0184] A bioreactor will typically be adapted to contain a liquid culture medium comprising the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph.

[0185] A bioreactor typically comprises mixing means adapted to mix the liquid culture medium and microorganisms contained therein. Some examples of mixing means include (but are not limited to) stirring or agitation means, and flow pumps. Alternatively, or additionally, gas flow may used to induce mixing, for example by bubbling a gas through a liquid culture medium.

[0186] A bioreactor will also typically be adapted to contact the liquid culture medium with a gas, such as the input gas or the intermediate gas. In order for the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph to make use of the input gas, at least part of the input gas (for example, methane, oxygen, and / or carbondioxide) must dissolve in the liquid culture medium. In some embodiments, a bioreactor may comprise means for increasing the rate of gas dissolution in the liquid culture medium.

[0187] In some embodiments, the bioreactor comprises mixing means adapted to mix the input gas and / or the intermediate gas with the liquid culture medium. In some embodiments, a mixing means is adapted to both mix the liquid culture medium and microorganisms contained therein, and also to mix the input gas and / or intermediate gas with the liquid culture medium. A variety of configurations are known in the art for such mixing, including (but not limited to) stirring or agitating the liquid culture medium in the presence of the gas, bubbling the gas through the liquid culture medium, or flowing the liquid culture medium in the presence of the gas. Various means may be used to increase the rate of gas transfer between the gas phase and the culture medium, such as increasing pressure, decreasing bubble size, altering the impeller type, or using baffles. Alternatively, or additionally, gas dissolution may be increased by providing a large contact surface between the liquid culture medium and the gas.

[0188] In some embodiments, the culture medium is agitated. The skilled person will be able to select an appropriate agitation rate, depending on the specifics of the system used. Possible mechanisms of agitation include the use of an agitator (such as a stirring impeller), pumping culture medium, and / or bubbling gas. Increased gas dissolution may generally be achieved by increasing the agitation rate, however this may also impart an increased shear force on the microorganisms when using certain types of agitator. In these cases, the increase in agitation may need to be balanced against an undesirable increase in cellular damage due to shear forces.

[0189] In some embodiments, the input gas and / or the intermediate gas is supplied (for example, bubbled through the culture medium) at a rate of at least about 5 mL per litre of culture medium per minute, such as at least about 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or at least about 350 mL per litre of culture medium per minute, and useful ranges may be selected between any of these values (for example, from about 5 to about 350, from about 5 to about 200, from about 5 to about 100, from about 5 to about 50, from about 5 to about 30, from about 5 to about 20, from about 8 to about 350, from about 8 to about 200, from about 8 to about 100, from about 8 to about 50, from about 8 to about 30, from about 8 to about 20, from about 12 to about 350, from about 12 to about 200, from about 12 to about 100, from about 12 to about 50, from about 12 to about 30, from about 12 to about 20, from about 16 to about 350, from about 16 to about 200, from about 16 to about 100, from about 16 to about 50, from about 16 to about 30, or from about 16 to about 20 mL per litre of culture medium per minute).

[0190] A bioreactor will also typically comprise one or more sensors such as (but not limited to) temperature sensor(s), pH sensor(s), pressure sensor(s), gas sensor(s) for determining the concentration of gases in the headspace, dissolved gas sensor(s) for determining the concentration of dissolved gases in the liquid culture medium, and / or light sensor(s).

[0191] A bioreactor will typically comprise regulation means for regulating one or more parameters, such as temperature, pH, pressure, gas composition, dissolved gas concentration, light intensity, and so on. These may be adapted to receive input from one or more sensors, thereby allowing the parameter(s) to be maintained within a range suitable for the growth of the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph. Such sensors and regulation means are well known in the art.

[0192] In some embodiments, a bioreactor may comprise lighting means such that the one or more strains of phototroph may be exposed to light. Alternatively, or additionally, a bioreactor may be adapted to allow the one or more strains of phototroph to be exposed to an external source of light, such as sunlight. One exemplary bioreactor comprising means for exposing one or more strains of phototroph to light is the tubular photobioreactor (Smith, 2020, "Optimisation of eicosapentaenoic acid productivity from a New Zealand microalga in a tubular photobioreactor", doctoral thesis, University of Canterbury, doi: 10.26021 / 10197).

[0193] Various lighting means are known in the art, including (but not limited to) incandescent lights, fluorescent lights, vapour lamps, and light-emitting diodes (LEDs). Preferred lighting means are warm-white LEDs.

[0194] In some embodiments, the lighting means are inside the bioreactor, for example in direct contact with the liquid culture medium, or separated from the liquid culture medium by a light-permeable material. In other embodiments, the lighting means are external to the bioreactor, and at least one portion of the bioreactor is permeable to light, thereby allowing penetration of light to the interior of the bioreactor. In such embodiments, the at least one portion of the bioreactor comprises a material that is permeable to light, preferably permeable to the wavelengths of light involved in photosynthesis. These wavelengths include light falling within the blue (425-450 nm) and red (600-700 nm) ranges. Glass and various plastics, such as acrylics (polymethlamethacrylate), butyrate (cellulose acetate butyrate), lexan (polycarbonate), PETG (glycol modified polyethylene terphthalate), and combinations thereof are impermeable to fluids and are permeable to visible light, making them suitable materials.

[0195] The skilled person will be able to select an appropriate intensity and wavelength of light, depending on the requirements of the particular strain(s) of phototroph and thebioreactor used. Light intensity may be measured using means known in the art, for example using a LI-250A Light Meter (LI-COR Biosciences). Preferably, light intensity is measured at the centre of the bioreactor in which the phototroph is to be cultured.

[0196] In some embodiments, when in use, the light intensity at the centre of the bioreactor is at least about 30 pmol photons m’2s’1, such as at least about 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 450, 500, 550, or at least about 600 pmol photons m’2s’1, and useful ranges may be selected between any of these values (for example, from about 30 to about 600, from about 30 to about 500, from about 30 to about 400, from about 30 to about 300, from about 60 to about 600, from about 60 to about 500, from about 60 to about 400, or from about 60 to about 300 pmol photons m’2s’1).

[0197] In some embodiments, the methods and / or systems may utilise a single-stage bioreactor design. For example, the methods and / or systems of the invention may comprise coculturing the one or more strains of methanotrophic bacteria and the one or more strains of phototroph together in a bioreactor.

[0198] Alternatively, the methods and / or systems may utilise a design comprising two or more bioreactors. In such embodiments, a first bioreactor may be adapted for culturing the one or more strains of methanotrophic bacteria, and a second bioreactor may be adapted for culturing the one or more strains of phototroph, or vice versa. Of course, it is also possible to utilise designs comprising additional bioreactors, and such additional bioreactors may be adapted to culture and / or coculture the same or different strains of methanotrophic bacteria and / or phototroph.

[0199] In methods and / or systems comprising two or more bioreactors, the first bioreactor will be adapted to receive the input gas and provide an intermediate gas, and the second bioreactor will be adapted to receive the intermediate gas and provide an output gas. Such systems thereby have a shared gas flow, in which the intermediate gas produced by the first bioreactor is provided as an input to the second bioreactor. This shared gas flow may be sequential (i.e. linear) or recirculated. For example, in some embodiments, the gas flows from the first bioreactor to the second bioreactor and then to the output; in other embodiments, at least a portion of the output gas is combined with the input gas and reintroduced to the first bioreactor.

[0200] Accordingly, in some embodiments the system is adapted to recirculate at least a portion of the output gas, and wherein the system is adapted to culture and / or coculture the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph in the presence of the input and recirculated gases.

[0201] The system may be configured to recirculate the gas as desired. For example, output gas from a methanotrophic bacterial culture may be recirculated to a phototroph culture, or a coculture of methanotrophic bacteria and phototrophs. Alternatively, or additionally, output gas from a phototroph culture may be recirculated to a methanotrophic bacterial culture, or a coculture.

[0202] The amount or rate of recirculation may be selected according to need. In some embodiments, the recirculated gas is combined with the input gas prior to introduction to the culture and / or coculture. The ratio of input gas to recirculated gas may therefore be varied as needed to adjust the composition of the gas mixture.

[0203] Without wishing to be bound by theory, it is believed that recirculating gas in this manner may allow a greater decrease in carbon containing gas and / or a greater production of biomass to be achieved.

[0204] Which microorganisms are cultured in the first and second bioreactors may be selected based on the composition of the input gas and the desired output. For example, when the input gas comprises low concentrations of methane (for example, less than about 5% v / v) and high concentrations of carbon dioxide (for example, at least about 70% v / v), it may be preferable for the first bioreactor to comprise the one or more strains of phototroph and the second bioreactor to comprise the one or more strains of methanotrophic bacteria. In this manner, the intermediate gas (i.e. the gas exiting the first bioreactor and entering the second bioreactor) may be at least partially depleted in carbon dioxide, and therefore proportionally enriched in methane, before entering the bioreactor comprising the methanotrophs.

[0205] The parameters of each bioreactor (for example, bioreactor size, shape, design, and / or material; temperature; culture medium composition and / or pH; gas composition and / or pressure; dissolved gas concentration, such as dissolved oxygen concentration; type and / or intensity of mixing; and / or light intensity, wavelength, and / or source type) may be optimised for the particular microorganism being cultured. This will be particularly useful when the one or more strains of methanotrophic bacteria and the one or more strains of phototroph have disparate growth requirements. In particular, the bioreactor for culturing the one or more strains of phototroph should allow for the phototroph to be cultured in the presence of light.8. Biomass

[0206] Biomass may include a wide variety of organic and inorganic compounds. In some embodiments, the biomass comprises:a) one or more biopolymers, preferably one or more proteins, glycoproteins, polypeptides, polysaccharides, and / or polynucleotides, b) one or more amino acids, c) one or more nucleotides and / or nucleosides, d) one or more carbohydrates, e) one or more lipids, f) one or more vitamins, g) one or more bioactive compounds, preferably one or more pigmentcontaining compounds, more preferably phycocyanin, or h) any combination of any two or more of (a) to (g).

[0207] It will be appreciated that the exact composition of the biomass will depend on a variety of factors, including the species and strain of methanotrophic bacteria and phototroph used, and the culture conditions.

[0208] In some embodiments, the biomass comprises and / or is produced by the methanotrophic bacteria. In some embodiments, the biomass comprises and / or is produced by the phototroph. In some embodiments, the biomass comprises and / or is produced by both the methanotrophic bacteria and the phototroph. This may occur when the methanotrophic bacteria and the phototroph are cocultured, or when they are cultured separately (for example, sequentially). For clarity, in embodiments where the methanotrophic bacteria and phototroph are cultured separately (for example, in two or more bioreactors), it is envisioned that the biomass may comprise and / or be produced by the methanotrophic bacteria, the phototroph, or by both the methanotrophic bacteria and the phototroph.

[0209] In some embodiments, the biomass comprises the methanotrophic bacteria and / or the phototroph. In some embodiments, the biomass comprises derivatives of the methanotrophic bacteria and / or the phototroph, for example killed and / or lysed bacteria and / or phototroph. In some embodiments, the biomass comprises one or more metabolic products of the methanotrophic bacteria and / or the phototroph.

[0210] Biomass may be harvested from cultures and / or cocultures by any suitable means known in the art. This will typically involve removal of a liquid phase. For example, harvesting biomass may comprise centrifuging a liquid culture and removing the liquid phase in order to recover the biomass in the solid phase. Alternatively, or additionally, harvesting biomass may comprise one or more steps such as filtration, spray-drying, and / or freeze- drying.

[0211] In some embodiments, the biomass comprises one or more biopolymers, preferably one or more proteins. In some embodiments, the biomass comprises at least about 1% w / w protein by dry weight, such as at least about 5%, 10%, 15%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 68%, or at least about 70% w / w protein by dry weight, and useful ranges may be selected between any of these values (for example, from about 1% to about 70%, from about 1% to about 60%, from about 1% to about 40%, from about 1% to about 20%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 40%, from about 10% to about 20%, from about 20% to about 70%, from about 20% to about 60%, from about 20% to about 58%, from about 20% to about 56%, from about 20% to about 54%, from about 20% to about 52%, from about 20% to about 50%, from about 30% to about 70%, from about 30% to about 60%, from about 30% to about 58%, from about 30% to about 56%, from about 30% to about 54%, from about 30% to about 52%, from about 30% to about 50%, from about 40% to about 70%, from about 40% to about 60%, from about 40% to about 58%, from about 40% to about 56%, from about 40% to about 54%, from about 40% to about 52%, from about 40% to about 50%, from about 50% to about 70%, or from about 50% to about 60%).

[0212] The nutritional value of a protein feed is largely determined by its amino acid profile, in particular the content of essential amino acids (i.e. amino acids that cannot be synthesised by humans or animals). The essential amino acids are histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. The non- essential amino acids are typically considered to be cysteine, tyrosine, glycine, glutamic acid, serine, aspartic acid, arginine, taurine, proline, and alanine, however arginine, cysteine, glutamine, glycine, proline, and tyrosine may also be referred to as conditionally essential amino acids, as they can be synthesised by humans, but certain populations (such as newborn infants or people with liver damage) may not be able to synthesise them in sufficient quantity, and they therefore must obtain at least some of these in their diet.

[0213] One method of describing the nutritional value of a protein feed is by reference to the FAO / WHO / UNU standard (WHO / FAO / UNU Expert Consultation, 2007, 'Protein and amino acid requirements in human nutrition.' World Health Organ Tech Rep. Ser 935:1-265). This standard gives the required daily intake of specific amino acids by humans, and a protein feed may therefore be evaluated by whether it would, if consumed as the sole dietary protein source at the recommended protein intake level of 0.66 g protein kg-1day-1, provide all the required essential amino acids for an adult. A hypothetical protein that provides the required daily intake of essential amino acids for an adult when consumed as the sole dietary protein source at the recommended protein intake level is referred to as the WHO / FAO / UNU standard.

[0214] The amino acid content of several reference proteins is given in Table 1.Table 1. Amino acid content of reference proteins.Amino acid contents are given as g / 100g protein.1Overland et aL, 2010, 'Evaluation of methane-utilising bacteria products as feed ingredients for monogastric animals', Archives of Animal Nutrition, 64(3), 171 -189, doi: 10.1080 / 174503910036915342Schultz et aL, 2006, 'Microbial production of single-cell protein from deproteinized whey concentrates', Applied Microbiology and Biotechnology, 69 5), 515-520. doi:10.1007 / s00253- 005-0012-z3Becker, 2007, 'Micro-algae as a source of protein', Biotechnology Advances, 25(2), 207-210. doi: 10.1016 / j.biotechadv.2006.11.0024World Health Organization (2007), 'Protein and amino acid requirements in human nutrition' (Vol. 935).5Total of all amino acids listed excluding Cys and Tyr.6Total of all amino acids listed.

[0215] In some embodiments, the biomass has an essential amino acid concentration of at least about 6 g / 100g dry weight, such as at least about 8g / 100g dry weight, at least about 10 g / 100g dry weight, at least about 12 g / 100g dry weight, at least about 14 g / 100g dry weight, at least about 16 g / 100g dry weight, at least about 18 g / 100g dry weight, at leastabout 20 g / 100g dry weight, at least about 22 g / 100g dry weight, at least about 24 g / 100g dry weight, or at least about 26 g / 100g dry weight, and useful ranges may be selected between any of these values (for example, from about 6 to about 26 g / 100g dry weight, from about 6 to about 24 g / 100g dry weight, from about 6 to about 22 g / 100g dry weight, from about 6 to about 20 g / 100g dry weight, from about 8 to about 26 g / 100g dry weight, from about 8 to about 24 g / 100g dry weight, from about 8 to about 22 g / 100g dry weight, from about 8 to about 20 g / 100g dry weight, from about 10 to about 26 g / 100g dry weight, from about 10 to about 24 g / 100g dry weight, from about 10 to about 22 g / 100g dry weight, from about 10 to about 20 g / 100g dry weight, from about 12 to about 26 g / 100g dry weight, from about 12 to about 24 g / 100g dry weight, from about 12 to about 22 g / 100g dry weight, from about 12 to about 20 g / 100g dry weight, from about 14 to about 26 g / 100g dry weight, from about 14 to about 24 g / 100g dry weight, from about 14 to about 22 g / 100g dry weight, from about 14 to about 20 g / 100g dry weight, from about 16 to about 26 g / 100g dry weight, from about 16 to about 24 g / 100g dry weight, from about 16 to about 22 g / 100g dry weight, from about 16 to about 20 g / 100g dry weight, from about 18 to about 26 g / 100g dry weight, from about 18 to about 24 g / 100g dry weight, from about 18 to about 22 g / 100g dry weight, or from about 18 to about 20 g / 100g dry weight).

[0216] In some embodiments, the biomass has an essential amino acid concentration of at least about 20 g / 100g protein, such as at least about 25 g / 100g protein, at least about 30 g / 100g protein, at least about 35 g / 100g protein, or at least about 40 g / 100g protein, and useful ranges may be selected between any of these values (for example, from about 20 to about 40 g / 100g protein, from about 20 to about 35 g / 100g protein, from about 25 to about 40 g / 100g protein, from about 25 to about 35 g / 100g protein, from about 30 to about 40 g / 100g protein, or from about 30 to about 35 g / 100g protein).

[0217] In some embodiments, the biomass comprises at least about 2.5 g lysine per 100 g protein, such as at least about 3.0, 3.5, 4.0, 4.5, 5.0, or at least about 5.5 g lysine per 100 g protein.

[0218] In some embodiments, the biomass comprises at least about 1.0 g methionine per 100 g protein, such as at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, or at least about 2.2 g methionine per 100 g protein.

[0219] In some embodiments, the biomass comprises at least about 0.4 g cysteine per 100 g protein, such as at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, or at least about 1.7 g cysteine per 100 g protein.

[0220] In some embodiments, the biomass meets or exceeds the WHO / FAO / UNU standard for at least 1 essential amino acid, such as at least 2, 3, 4, 5, 6, 7, or at least 8essential amino acids. In some embodiments, the biomass comprises protein that, if consumed as the sole dietary protein source at 0.66 g protein kg-1day-1, would meet or exceed the required daily human intake for: a) isoleucine, b) leucine, c) lysine, d) methionine, e) phenylalanine, f) threonine, g) tryptophan, h) valine, or i) any combination of any two or more of (a) to (h).

[0221] In some embodiments, the biomass comprises one or more carbohydrates. In some embodiments, the biomass comprises at least about 10% w / w carbohydrate by dry weight, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or at least about 70% w / w carbohydrate by dry weight, and useful ranges may be selected between any of these values (for example, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 20% to about 70%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 30% to about 70%, from about 30% to about 60%, from about 30% to about 50%, or from about 30% to about 40%).

[0222] In some embodiments, the biomass comprises one or more polysaccharides, preferably glycogen. Glycogen is a branched polysaccharide of glucose that is used by many organisms for energy storage. Glycogen can be used in a variety of applications known in the art including as a replacement for amylopectin or starch in nutritional and medical compositions.

[0223] In some embodiments, the biomass comprises at least about 10% w / w polysaccharide, such as glycogen, by dry weight, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or at least about 70% w / w carbohydrate, such as glycogen, by dry weight, and useful ranges may be selected between any of these values (forexample, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 20% to about 70%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 30% to about 70%, from about 30% to about 60%, from about 30% to about 50%, or from about 30% to about 40%).

[0224] In some embodiments, the biomass comprises one or more bioactive compounds, preferably one or more pigment-containing compounds, more preferably phycocyanin. Many microbes such as those anticipated for use in the present invention are known to produce a number of useful bioactive compounds, often as secondary metabolites. For example, phototrophs will typically produce a variety of pigment-containing compounds, such phycocyanin, as part of the biochemical pathway for harvesting light energy and converting it into chemical energy.

[0225] Phycocyanin is produced by a number of cyanobacteria and microalgae, and has excellent antioxidant and anti-inflammatory properties. In some embodiments, the biomass comprises phycocyanin. It is anticipated that a wide variety of bioactive compounds may be present in such biomass, and the nature of these compounds will depend on the specific microbes used.

[0226] It is envisioned that the biomass described herein may be useful for a variety of purposes including (but not limited to) as a component of a nutritional composition such as a human nutritional composition or an animal feed composition, or as a plant fertiliser.9. Nutritional compositions

[0227] In some embodiments, biomass produced using the methods and / or systems of the invention may be useful as a component of a nutritional composition. In some embodiments, a nutritional composition may comprise, consist essentially of, or consist of biomass produced using the methods and / or systems of the invention.

[0228] The biomass described herein has a high protein content, making it particularly useful to increase the protein content of feed compositions that would otherwise contain relatively low levels of protein. It is also envisioned that biomass may be useful to supplement particular amino acids (for example essential amino acids, and in particular lysine, methionine, and / or cysteine) that may be deficient in other protein sources.

[0229] In some embodiments, the nutritional composition is a human nutritional composition. In some embodiments, the nutritional composition is an animal feed composition.

[0230] The term "human nutritional composition" means a nutritional composition intended for consumption by humans. Human nutritional compositions include (but are not limited to) food compositions, beverage compositions, and food or beverage ingredient compositions.

[0231] In some embodiments, the human nutritional composition comprises a protein powder, a protein shake, a protein shot, a protein gel, a sports nutritional formulation, a UHT beverage, a UHT smoothie, a sauce, a spread, a jam, a jelly, an acid-set gel, a yoghurt, a neutral beverage, an acidic beverage, a water, a juice, a milk, a smoothie, a shake, a shot, an alcoholic beverage, a soft drink, a kombucha, a kefir, a ready-to-mix powder, a bar, a ball, a cake, a cookie, a muffin, or a bakery good, a medical food, a soup, a dessert, a pudding, a custard, an enteral formulation, a formulation for senior or aged populations, a tablet, a capsule, or a supplement, a confection, a gummy, a candy, a chocolate, a fudge, a truffle, a chewing gum, a frozen dessert, an ice cream, flavouring, a topping, or a baking ingredient, a cream, a cheese, or a butter.

[0232] In one embodiment, the human nutritional composition comprises a bakery good. In some embodiments, the bakery good is a bread, a bar, a ball, a cake, a cookie, or a muffin.

[0233] In some embodiments, the human nutritional composition comprises a beverage. In some embodiments the beverage is a protein shake, a protein shot, a UHT beverage, a UHT smoothie, a neutral beverage, an acidic beverage, a water, a juice, a milk, a smoothie, a shake, a shot, an alcoholic beverage, a soft drink, a fermented drink, a kombucha, a kefir, a ready-to-drink (RTD) beverage, a coffee beverage, a tea, or a creamer. In some embodiments, the human nutritional composition comprises a coffee capsule, an instant coffee, a tea bag, or a coffee creamer.

[0234] In one embodiment, the human nutritional composition comprises a breakfast food. In some embodiments, the breakfast food is a cereal, oats, a porridge, a spread, a jam, a jelly, or a honey.

[0235] In one embodiment, the human nutritional composition comprises a confectionary. In some embodiments, the confectionary is a boiled sweet, a candy, a chewing gum, a chocolate bar, a chocolate-coated product, a confectionary bar, a fudge, or a truffle.

[0236] In one embodiment, the human nutritional composition comprises a dairy product. In one embodiment, the dairy product is a yoghurt. In some embodiments, the yoghurt is a chilled yoghurt, an ambient yoghurt, a stirred yoghurt, a set yoghurt, or a drinking yoghurt. In one embodiment, the dairy product is a milk. In some embodiments, themilk is fresh milk, UHT milk, fortified milk, flavoured milk, or a fermented milk product such as kefir. In one embodiment, the dairy product is a cheese. In some embodiments the cheese is a block cheese, a cream cheese, or a processed cheese. In some embodiments the dairy product is a cream, such as a fresh cream or a UHT cream. In some embodiments, the dairy product is an ice cream.

[0237] In one embodiment, the human nutritional composition comprises a dessert. In some embodiments, the dessert is an ice cream, a sorbet, a frozen yoghurt, a pudding, a mousse, a jelly, a fruit puree, or a dessert topping.

[0238] In one embodiment, the human nutritional composition is a meal ingredient. In some embodiments the meal ingredient is a condiment, congee (rice porridge), a foam cream, instant noodles, an oil, a pasta, a rice, a salad dressing, or a seasoning.

[0239] In one embodiment, the human nutritional composition comprises a medical food or beverage. In some embodiments, the medical food or beverage is a meal replacement, a meal replacement shake, a nutritional drink, a soup, a pudding, a nutritional powder, a liquid diet, or a dietary supplement. In some embodiments, the medical food or beverage comprises an encapsulated probiotic and / or an antibiotic.

[0240] In one embodiment, the human nutritional composition comprises a powder. In some embodiments the powder is a protein powder, a ready-to-mix beverage powder, or a meal replacement powder.

[0241] In one embodiment, the human nutritional composition comprises a snack. In some embodiments, the snack is a baked good, a cheese lollipop, chips, a bar, a cookie, a dip, dried fruit, nuts, a ball, a fruit leather, a high protein baked good, a yoghurt drop, a grain mix, popcorn, potato chips, a smoothie bar, or a veggie chip.

[0242] In some embodiments the animal feed composition is a feed composition for farmed animals. In some embodiments, the animal feed composition is a ruminant feed composition. Animal feed compositions suitable for ruminants are described in E. W. Crampton et al., Applied Animal Nutrition, W. H. Freeman and Company, San Francisco, CA., 1969 and D. C. Church, Livestock Feeds and Feeding, 0 & B Books, Corvallis, Oreg., 1977, both of which are incorporated herein by reference. In some embodiments, the ruminant feed composition is a feed composition for bovines, such as dairy cows and / or beef cows.

[0243] In some embodiments, the animal feed composition is a pet food composition for companion animals such as cats and dogs. In certain embodiments, the pet food is a dog food. In certain embodiments, the pet food is a cat food. The terms "pet food", or "pet food composition" as used herein means nutritional compositions intended for ingestion by a pet.In one embodiment a nutritional composition may refer to a dietary supplement intended for ingestion by a pet. A dietary supplement is intended to refer to a composition that provides nutrients that may otherwise not be consumed in sufficient quantities by the pet. In one embodiment a nutritional composition may refer to a pet treat intended for ingestion by a pet. The term "pet treat" as used herein refers to a food for consumption by a pet that is intended as an occasional reward or indulgence and not as the sole source of a pet's nutrition.

[0244] In some embodiments, the animal feed composition is a livestock food, a pet food, a kibble, pet biscuit, a pet treat, an animal feed ingredient, a livestock forage, a livestock supplement, or a drench.

[0245] In some embodiments, the animal feed composition is a feed composition for omnivores such as chickens, pigs, and dogs. Such feed compositions are well known in the art.

[0246] In some embodiments, the animal feed composition is a composition for aquatic animals, for example marine animals, for example a fish feed composition.

[0247] Animal feed compositions typically include a number of different components, depending on the nutritional requirements of the animal(s) in question. Such additional components will be well known to those skilled in the art, and may include sources of proteins, carbohydrates, fats, fibre, vitamins, minerals, and the like. In some embodiments, an animal feed composition may comprise one or more components in addition to the biomass produced using the methods and / or systems of the invention.

[0248] Examples of animal feed compositions and other components of animal feed compositions include Partial or Total Mixed Ration (TMR), corn, soybean, forage, grain, distiller's grain, sprouted grain, legumes, vitamins, amino acids, minerals, fibre, fodder, grass, hay, straw, silage, kernel, leaves, meal, solubles, slurries, supplements, mash feed, meal, fruit pulp, vegetable pulp, fruit or vegetable pomace, citrus meal, wheat shorts, corn cob meal, and molasses, milk, milk powder, milk replacement, milk fortifier, colostrum, whey, whey powder, sucrose, maltodextrin, rice hulls and the like.

[0249] An animal feed composition may be formulated as a food, drink, food additive, drink additive, total animal feed, animal feed additive, animal feed supplement, dietary supplement, carrier, vitamin or mineral premix, nutritional product, enteral feeding product, soluble, slurry, supplement, pharmaceutical, lick block, drench, tablet, capsule, pellet or intra- ruminal product, e.g., a bolus. Appropriate formulations may be prepared by an art skilled worker with regard to that skill and the teaching of this specification.

[0250] Animal feed compositions may be administered as a top dressing on, or mixed into, a standard feed material such as a daily ration. In addition, biomass be administered in a partial or total mixed ration (TMR), pelleted feedstuff, mixed in with liquid feed or drink, mixed in a protein premix, or delivered via a vitamin and mineral premix. EXAMPLES1. Example 1 — Stable coculture in batch formatMaterials and methods

[0251] A modified V4 medium (Dunfield et aL, 2007, "Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia", Nature, 450(7171), 879-882, doi: 10.1038 / nature06411) was used for all cultures. The modified V4 medium was prepared by dissolving the components shown in Table 2 in 1 L of deionised water and adjusting the pH to 2.5 with H2SO4. The FeEDTA solution was prepared by dissolving 1.54 g FeSO4-7H2O and 2.06 g Na2EDTA in 1 L of deionised water. Trace element solution 1 was prepared by dissolving the components shown in Table 2 in 1 L of deionised water. The trace metal solution was prepared by dissolving 1.5 g of nitrilotriacetic acid in 800 mL of deionised water, adjusting the pH to 6.5 with KOH, adding the remaining minerals shown in Table 2 in order, adjusting the pH to 7.0, and adding deionised water to bring the volume to 1 L.Table 2. Composition of modified V4 medium, trace element solution, and trace metal solution.

[0252] Cultures of Methylacidiphilum sp. RTK17.1 were maintained in chemostat using a 1 L bioreactor (BioFlo 110; New Brunswick Scientific) with a 600 mL working volume and 0.0069 h-1dilution rate. A gas mixture (69 % CO2, 1.0 % CH4, 3.1 % O2, balance N2, all v / v) was supplied at 20 mL min-1. Temperature was maintained at 50°C, agitation at 800 rpm, and pH ~ 2.5 (but not controlled). Under these conditions, typical ODeoo values were 1.2 A.U. and outlet gas concentrations were 72 % CO2, 0.13 % CH4, 1.5 % O2, with the remaining attributed to N2.

[0253] Cultures of Galdieria sp. RTK37.1 were maintained in batch format. 300 mL stocks were grown in batch cycles in 1 L pressure bottles with rubber stoppers. At the beginning of each cycle, the phototrophic microalgae were diluted to ODeoo 1.0 with fresh V4 media, the bottles were subjected to a vacuum for 3 minutes, and re-pressurized to 5 psia with an 80% v / v CO2 and 20% v / v N2 gas mixture. The bottles were incubated horizontally in a shaking incubator at 110 rpm and 45°C. Light was provided from the top at 60 pmol photons m’2s’1measured at bottle's level. Growth was stopped at ODeoo 6.0 A.U. and the cycle started anew. For all experiments, Galdieria sp. RTK37.1 was aseptically harvested at a ODeoo 5.0 A.U. and diluted with fresh V4 media to the desired starting concentration.

[0254] For batch coculture experiments, 250 mL of sterile V4 media was added to 1 L pressure bottles sealed with rubber stoppers. Galdieria sp. RTK37.1 was inoculated to a starting ODeoo of 0.9 A.U. and Methylacidiphilum sp. RTK17.1 to an ODeoo 0.1 A.U. The axenic Galdieria controls were inoculated to a starting ODeoo of 1.0 A.U. and the axenic Methylacidiphilum controls to ODeoo 0.1 A.U. Coculture headspaces were prepared by subjecting the bottle to vacuum for 3 minutes and then re-pressurizing to 5 psia with an 80% CO2 and 20% N2 gas mixture (v / v). The bottles were subjected to vacuum and repressurized 3 times. After this, headspace gas was equilibrated to atmospheric pressure by expanding against a syringe, and then 120 mL of 100% CH4 was injected. The headspace gas was then released to 10 kPa. The axenic Galdieria headspace was prepared in a similar fashion, butwithout CH4 addition. The headspace air was not replaced for the Methylacidiphilum controls, but 120 mL of CH4 and 60 mL of CO2 were injected, and then the pressure released to 10 kPa. The headspace composition is shown in Table 3. All cocultures and controls were run in duplicates.Table 3. Headspace gas concentrations for batch coculture experiments.1Balance is N2 for all cultures.

[0255] All bottles were incubated horizontally in a shaking incubator at 110 rpm and 45°C. Light was provided from the top at 60 pmol photons m-2s’1measured at bottle level. Liquid (2 mL) and gas (10 mL) samples were taken daily. To minimise glycogen accumulation under N-depletion, experiments were stopped when ammonium was depleted, or the stationary phase was reached. To harvest the biomass for nutritional analysis, the replicates were combined and centrifuged at 12,000 rpm for 15 minutes. Supernatants were discarded, and each biomass pellet was stored at -20°C until needed.

[0256] Biomass concentrations were estimated by measuring ODeooand using conversion factors obtained in axenic cultures. One unit of ODeoo was equivalent to 0.308 gow L’1for Galdieria sp. RTK37.1 and 0.435 gow L’1for Methylacidiphilum sp. RTK17.1. For cocultures, relative amounts of Methylacidiphilum and Galdieria were estimated using the oxygen uptake / production rate of the axenic cultures and the oxygen production of the coculture.

[0257] Gas samples were analysed for CO2, CH4, N2, and O2 concentrations using gas chromatography.

[0258] Ammonium concentrations were monitored as described using the orthophthaldialdehyde (OPA) method (Taylor et al., 1974, "Cathepsin B2 measurement by sensitive fluorometric ammonia analysis", Analytical Biochemistry, 60(1), 153-162, doi: 10.1016 / 0003-2697(74)90140-7) on liquid samples filtered through 0.2 pm nylon syringe filters.Results

[0259] To assess coculture viability and to study differences between coculture and axenic growth, Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 were cultivated within gastight bottles in batch mode.

[0260] Axenic methanotroph batch cultures reached stationary phase following 4 days incubation with an ODeoo of 0.40 ± 0.03 A.U. The onset of stationary phase corresponded to an O2 limitation in the headspace (0.9 % O2; Figure 1a). Galdieria sp. RTK37.1 cultures and cocultures showed similar growth to each other, with final ODeoo values at day 4 of 3.95 ± 0.07 A.U. and 4.05 ± 0.21 A.U. respectively.

[0261] The specific growth rate for Methylacidiphilum sp. RTK17.1 in coculture (0.022 h"1) was significantly faster than in the axenic control (0.017 h’1; p-value < 0.05).

[0262] The coculture showed enhanced CH4 removal compared to the axenic Methylacidiphilum culture (Figure 1 b). In the axenic Galdieria culture, O2 steadily accumulated, whereas in the axenic Methylacidiphilum culture it steadily depleted (Figure 1a). In the coculture, the O2 concentration remained stable (between 1.4-3.4 % v / v) until CH4 was depleted on day 3 (Figure 1 b) at which point it rapidly increased.

[0263] CO2 is required for, and consumed by, photoautotrophic growth of Galdieria; and is produced during CH4 oxidation by Methylacidiphilum. In axenic Methylacidiphilum culture, CO2 steadily increased; in axenic Galdieria culture it steadily decreased; and in coculture, CO2 remained relatively stable until CH4 was depleted on day 3 (Figure 1c). Neither the axenic Galdieria, nor the coculture showed signs of CO2 inhibition.

[0264] Methane is the energy source for Methylacidiphilum sp. RTK17.1, so its consumption is necessary for the methanotroph to grow. In the axenic Methylacidiphilum culture, CH4 was slowly consumed until day 4, when O2 reached 0.9 % v / v, with 49.5 % of methane removed. No further growth or change in gas concentrations was observed in the subsequent days (data not shown). In contrast, in the coculture, CH4 oxidation was enhanced by the continuous O2 production from Galdieria. CH4 was completely removed from the headspace (LDL < 0.01 % CH4) within 3 days incubation. As < 2 % residual O2 was initially present in the coculture, the stoichiometric O2 requirement for CH4 oxidation dictates that both Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 were metabolically active in the coculture. Furthermore, in coculture CH4 was completely removed without an explosive gas mixture (as defined by Janes et al., 2011, "Experimental study of CH4 / O2 / CO2 mixtures flammability", 11 AIChE - 2011 AIChE Spring Meeting and 7th Global Congress on Process Safety, Conference Proceedings.) being present.

[0265] To better understand the differences in behaviour between the coculture and the axenic cultures, production and consumption rates were derived (Figure 2). The Methylacidiphilum axenic culture and the coculture both showed an increasing O2 consumption rate (i.e. negative O2 production) with time, until day 3 when CH4 and O2 limitations affected the coculture and methanotroph respectively. There was a small net O2 production (Figure 2a) at the start of coculture, which decreased to a net consumption by day 3, as CH4 oxidation consumed most of the O2 produced by the Galdieria. More O2 was produced by the axenic Galdieria than what was consumed by the axenic Methylacidiphilum.

[0266] When the O2 headspace concentration dropped to 0.9% (shortly after day 3) the O2 production rates for coculture and axenic Galdieria equated (p-value > 0.05). While O2 production / consumption was balanced in the coculture as long as there was methanotrophic activity, a modest net CO2 (Figure 2c) fixation was observed. Thus, a proportion of the CO2 produced during CH4 oxidation in coculture is assimilated into both new Methylacidiphilum and Galdieria biomass.

[0267] Methane consumption rates within axenic Methylacidiphilum cultures (Figure 2b) slowed following the first day, and ceased after day 4. In contrast, in cocultures CH4 oxidation rates increased continuously until CH4 was depleted shortly after day 3 (the apparent decrease between days 3 and 4 is a consequence of CH4 depletion). This enhanced rate of CH4 oxidation occurred even at headspace O2 concentrations that stopped CH4 oxidation in the axenic Methylacidiphilum culture.

[0268] With the exception of the first and last day of culture (when CH4 oxidation stopped due to O2 or CH4 depletion), CH4 consumption rates were 4-fold faster in the coculture than in the Methylacidiphilum axenic control (p-value < 0.001). Ammonium consumption rates (Figure 2d) were steady for Methylacidiphilum cultures and relatively slow when compared to Galdieria and coculture experiments, thus the presence of the methanotroph does not significantly impact the coculture's ammonium uptake (p-value > 0.05).2. Example 2 — Stable coculture in chemostat formatMaterials and methods

[0269] Cultures of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 were maintained as described in Example 1.

[0270] Continuous reactors were set up for axenic cultures of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1, and coculture. For each, a 1 L bioreactor (BioFlo 110, New Brunswick Scientific) was used, cultures were incubated at 45°C and V4 media at pH 2.5 wasused as described in Example 1. Bioreactor working volume was maintained at 900 mL with a scavenging / effluent pump. The axenic Methylacidiphilum culture and coculture were maintained at 400 rpm agitation, and fresh media was supplied, and spent media withdrawn, at a constant flow rate of 150 mL day-1(D= 0.167 day-1). The axenic Galdieria culture was maintained at 250 rpm agitation, and fresh media was supplied, and spent media withdrawn, at a rate of 250 ml day-1(D = 0.278 day-1).

[0271] Custom gas mixtures were supplied at 16 ml min1and were filter sterilised before entering the reactors. Feed gas composition for the axenic Methylacidiphilum culture and the coculture were approximately 2.1 % O2, 1.1 % CH4, 74 % CO2, balance N2, (all v / v). For the axenic Galdieria culture, feed gas composition was 3.0 % O2, 64 % CO2, balance N2 (all v / v). Light was supplied to the axenic Galdieria culture and coculture at 100 pmol photons m"2s’1measured at the centre of the empty reactor.

[0272] To initiate axenic cultures, reactors were inoculated to an ODeoo of 0.1 A.U. with the corresponding microorganism and grown in batch mode until an ODeoo of 1.0 A.U. was reached; after which the inlet and outlet peristaltic pumps were activated. For the coculture, the reactor was inoculated to an 0.1 ODeoo of Galdieria sp. RTK37.1, grown in batch until ODeoo = 1.0 A.U. and then the pumps were started. After achieving steady state, < 1 mL Methylacidiphilum sp. RTK17.1 (ODeoo = 1.20 A.U.) was added to the reactor. Liquid (2 mL) and outlet gas (40 mL) samples were harvested daily. Systems were considered at steady state when there was less than a 5 % ODeoo change over a five day period.Results

[0273] Chemostat cocultures of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 were compared with individual axenic cultures (Table 4). Steady state was achieved in all three cultures.Table 4. Steady state values in axenic cultures and cocultures of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 during chemostat cultivation.Values in brackets represent one standard deviation.a12 day average after steady state was achieved.bBiomass concentration of accumulated one day outflow broth, with 3 technical replicates.

[0274] The presence of Galdieria sp. RTK37.1 in the coculture increased the amount of dissolved oxygen in the broth, and decreased volumetric O2 net consumption by 46%, when compared to the axenic Methylacidiphilum. In this instance, the Galdieria was unable to completely support the oxygen demand by the methanotroph, as indicated by the negative value for net O2 production. This is likely due to a lower ratio of Galdieria:Methylacidiphilum in this experiment compared to the batch coculture of Example 1 Galdieria:Methylacidiphilum ratios of ~ 1.3 and -3.6 respectively). As a result, the chemostat coculture required at least some external oxygen supplementation.

[0275] In the coculture chemostat, a biomass concentration of 0.393 gow L and a biomass productivity of 2.74 mg L’1h-1were achieved, both greater than the respective axenic cultures values. The biomass yield on methane in the Methylacidiphilum reactor was 4.89 ± 0.17 gow molcH41, whereas in the coculture, the yield increased to 13.0 ± 0.9 gow mo1.

[0276] The methane consumption rate was lower in coculture than the axenic Methylacidiphilum culture, however this was likely an artefact due to the low agitation rate used (250 rpm used in this example vs 800 rpm used for culture maintenance). The reduction in agitation rate resulted in a decrease in Methylacidiphilum ODeoo from 0.92 to 0.29, and CH4 removal from 87.2% to 40.5%. Increasing methane mass transfer has a major effect on biomass concentration, yields, and growth rates of methanotrophs.

[0277] The beneficial effect Galdieria exhibited on CH4 oxidation in Example 1 was not observed in chemostat culture. This is likely due to O2 being continuously supplied in the chemostat culture. As the Methylacidiphilum in the coculture did not require additional O2 (because sufficient O2 was supplied by the feed gas), it obtained no benefit from the oxygenic activity of Galdieria.3. Example 3 — Nutritional analysis of biomassMaterials and methods

[0278] Cultures of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 were maintained as described in Example 1. Batch cultures and V4 medium were as described in Example 1 with nitrogen-replete (N-rep) cultures being harvested when ammonium wasdepleted or when stationary phase was reached, and nitrogen-deficient (N-def) cultures being allowed to reach stationary phase.

[0279] Chemostat cultures were as described in Example 2. The steady state outflow broth was collected daily and stored at 4°C. After 1 L of broth had accumulated, biomass was harvested by centrifugation, and the resulting biomass pellets were stored at -20°C until needed.

[0280] Nitrogen replete stirred tank reactor cultures (STR 10L, N-rep) of Methylacidiphilum sp. RTK17.1 were grown in 10 L stirred tank reactors (10 L Biostat D-DCU STR reactor; Sartorius, Germany) with static liquid and a continuous gas feed of 12.3% CO2, 2.5% CH4, 7.9% O2, balance N2, (all v / v) at 800 mL min-1. The temperature was maintained at 50°C with agitation at 500 rpm and pressure at 100 mbar. To avoid N-limitation, 4 g of NH4CI dissolved into 50 mL deionized water was added at ODeoo = 3.0 A.U. (120 h) and ODeoo = 6.0 A.U. (198 h). Biomass was harvested at 220 h (ODeoo = 9.2 A.U.) by centrifugation and stored at -20°C until needed.

[0281] Nitrogen deficient stirred tank reactor cultures (STR 1 L, N-def) of Methylacidiphilum sp. RTK17.1 were grown with static liquid and continuous gas feed as described for the nitrogen replete stirred tank reactor cultures, except that NH4CI was not added.

[0282] Photoreactor cultures of Galdieria sp. RTK37.1 were grown in an 80 L tubular photobioreactor (Smith, 2020, "Optimisation of eicosapentaenoic acid productivity from a New Zealand microalga in a tubular photobioreactor", doctoral thesis, University of Canterbury, doi: 10.26021 / 10197). Briefly, Galdieria sp. RTK37.1 were first cultivated in 250 mL baffled shake flasks. When Galdieria ODeoo reached 6.4 A.U., it was used to inoculate 1350 mL of sterile V4 media in a 1.5 L concentric tube airlift reactor. When OD reached 6.6 A.U., the entire contents of the airlift reactor were used to inoculate an 80 L tubular photobioreactor.

[0283] The 80 L tubular photobioreactor was as described in Smith (2020). Light intensity was set to 100 nmolphotons m-2 s’1, and then increased to 300 nmolphotons m-2 S’1after 5 days of culture. On day 19, prior to ammonium depletion (ODeoo = 3.6 A.U.), 24 L were harvested (photo, N-rep). In order to analyse N-depleted Galdieria sp. RTK37.1, the reactor was topped up to 76 L with reverse osmosis water. The culture was allowed to continue for an additional 7 days, and then harvested in its entirety on day 26 (ODeoo = 4.7 A.U.)(photo, N- def). Next, the harvested Galdieria (both N-replete and N-deficient) were separately vacuum filtered, and the collected biomass washed with deionised water. The washed biomass paste was scraped from the filter, collected, freeze-dried, and stored at -80°C until needed.

[0284] Nutritional characterization of biomass (ash, crude protein, fat, carbohydrates, and amino acid profile) was performed according to the official methods of analysis of the Association of Official Analytical Communities (AOAC, 2005) international. Ash content was determined by the furnace method (AOAC method 9442,05), total crude protein was determined via the Dumas method (AOAC method 968.06), fat content was determined by the Mojoinnier method (AOAC method 922.06), and carbohydrates were determined by difference. Amino acid profile determination of acid-stable residues was performed via reverse-phase high performance liquid chromatography (HPLC) separation using AccQ derivatization of biomass (60-140 mg) samples following oxidization with performic acid, and hydrolysis with hydrochloric acid as described in AOAC method 994.12 (AOAC, 2005). Cysteine and methionine content was determined by performic acid oxidation (AOAC method 985.28).Results

[0285] The macromolecular composition of cultures and cocultures of Methylacidiphilum sp. sp. RTK17.1 and Galdieria sp. RTK37.1 is shown in Table 5.able 5. Macromolecular composition of cultures and cocultures of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1.00286] The amino acid distribution of cultures and cocultures of Methylacidiphilum sp. sp. RTK17.1 and Galdieria sp. RTK37.1 is shown in able 6. able 6. Amino acid distribution for cultures and cocultures of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1.alues shown are g / 100gDw, and values in brackets are standard deviations with n = 3 technical replicates for all biomass samples, except TK37.1 (chemostat) with n = 2.

[0287] Biomass produced from the chemostat coculture contained 57.5% w / w protein, which compares favourably with protein sources used in animal feed such as soybean meal (44% w / w protein), and fishmeal (60% w / w protein; Elangovan & Shim, 2000, "The influence of replacing fish meal partially in the diet with soybean meal on growth and body composition of juvenile tin foil barb (Barbodes altus)", Aquaculture, 189(1), 133-144, doi: 10.1016 / S0044-8486(00)00365-3).

[0288] Biomass produced from the chemostat coculture had higher concentrations of tyrosine and cysteine that the axenic Methylacidiphilum culture (p-values < 0.001, and < 0.005 respectively), but lower concentrations than Galdieria culture (p-value < 0.05 for both amino acids). Biomass produced from the chemostat coculture had an essential amino acid concentration of 20.84 ± 0.90 g / 100gDw, and an essential amino acid concentration per 100g protein that is comparable to soybean meal and fishmeal (Figure 3; Overland et aL, 2010, "Evaluation of methane-utilising bacteria products as feed ingredients for monogastric animals", Archives of Animal Nutrition, 64(3), 171 -189, doi: 10.1080 / 17450391003691534).

[0289] The FAO / WHO / UNU standard states the required daily intake of specific amino acids by humans (WHO / FAO / UNU Expert Consultation, 2007), and is often used to evaluate the quality of a protein source. The protein biomass from coculture was comparable in quality to both fishmeal and soybean meal, and met the FAO / WHO / UNU standard for all indispensable amino acids, except that it was slightly deficient in histidine (Figure 3).4. Example 4 — Coculture ratiosMaterials and methods

[0290] Cultures of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 were maintained as described in Example 1.

[0291] Cocultures were conducted with varying initial Methylacidiphilunr.Galdieria mass ratios. Initial Galdieria sp. RTK37.1 concentrations were kept consistent at 0.215 gow L’1(0.88 A.U. ODeoo) with varying initial Methylacidiphilum sp. RTK17.1 concentrations, ranging between 0.0 to 0.52 gow L’1, (0.0 to 0.44 A.U. ODeoo). Coculture suspensions (40 mL) were prepared by aseptically diluting the appropriate stocks with sterile V4 media into 160 mL serum bottles. Sufficient coculture suspension was prepared to also measure the initial total biomass concentration gravimetrically. Each serum bottle was sealed and flushed for 3 minutes with an 80% CO2 and 20 % N2 gas mixture (v / v) and pressurized to 10 psia.

[0292] For each of the varying mass ratios, 6 serum bottles were prepared, into which half received 20 mL CH4 to allow growth of Methylacidiphilum sp. RTK17.1. N2 (20 mL) wasinjected into the remaining serum bottles to serve as a non-methanotroph growing control. These preparations resulted in initial headspace concentrations of approximately 0.5% O2, 13.0% CH4, 70% CO2, balance N2 (all v / v) for the CH4 containing cocultures, and 0.5% O2, 70% CO2, balance N2 (all v / v) for the non-CFU containing cocultures.

[0293] The serum bottles were then cultivated for 6 days on a shaking incubator at 45°C, 150 rpm, and light intensity of 20 molPhotons m’2s’1. Each day, 2 mL liquid samples were collected from each bottle and analysed for total ODeoo, pigment fluorescence, and absorption spectra measurements. At the end of the experiment, final gauge pressure was measured, liquid and gas samples were collected, and analysed for headspace gas concentration, and final biomass concentration.Results

[0294] To evaluate the influence of the mass ratio of Methylacidiphilum:Galdieria on coculture performance, several batch coculture experiments were performed with varying initial biomass ratios.

[0295] In the cocultures that did not receive CH4, Methylacidiphilum sp. RTK17.1 was unable to grow, and therefore increasing its initial concentration had minimal impact on the observed coculture growth rate (Figure 4a). Nevertheless, a small but significant decrease (9%, p-value < 0.005) in the overall growth rate was observed with increasing Methylacidiphilum sp. RTK17.1 proportional abundances, likely due to a shading effect.

[0296] In cocultures that did receive CH4, the growth rate and biomass productivity both dramatically decreased with increasing Methylacidiphilum:Galdieria mass ratios. In cocultures with mass ratios < 0.18, a small continuously slowing of coculture growth rate (8 % decrease, p-value < 0.01) was observed. Increasing the initial Methylacidiphilum concentration any further, however, slowed growth significantly (p-value < 0.001), with no growth observed at a mass ratio of 1.33 gow L’1: gow L’1. This result cannot be explained solely by cell shading, as the effect was not as intense in the cocultures without CH4. Without CH4, increasing Methylacidiphilum concentration 7.4-fold Methylacidiphilum:Galdieria ratio 0.18 to 1.33) slowed the overall growth rate by only 11 %; while in the CH4 containing cocultures, increasing Methylacidiphilum concentration 1.3-fold (mass ratio 0.18 to 0.23) slowed the overall growth rate by 66 % (Figure 4a).

[0297] For initial mass ratios > 0.23 (Methylacidiphilum Galdieria), much less biomass was produced when CH4 was available (Figure 4b), suggesting that Methylacidiphilum sp. RTK17.1 growth can impede Galdieria sp. RTK37.1 growth.

[0298] All cocultures grown with CFUat an initial Methylacidiphilum:Galdieria mass ratio > 0.23 developed a yellow coloration within 4 days incubation (not shown). In microalgae, N- starvation is known to trigger a similar colour change, known as 'chlorosis', which is caused by the loss of chlorophylls and phycobiliproteins (Salbitani & Carfagna, 2020, "Different behaviour between autotrophic and heterotrophic Galdieria sulphuraria (Rhodophyta) cells to nitrogen starvation and restoration. Impact on pigment and free amino acid contents", International Journal of Plant Biology, 11 (1), doi: 10.4081 / pb.2020.8567). However in this Example, excess ammonium was supplied to the cocultures, so the chlorosis and slow growth observed are not attributable to N-limitation. The pigment loss coincided with the appearance of coproporphyrin in solution (as evidenced by the appearance of an absorbance peak at 400 nm, data not shown). Coproporphyrin is an intermediate in the biosynthesis of both phycocyanin and chlorophyll (Stadnichuk et aL, 1998, 'Far-red light-regulated efficient energy transfer from phycobilisomes to photosystem I in the red microalga Galdieria sulphuraria and photosystems-related heterogeneity of phycobilisome population', Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1807(2), 227-235, doi:10.1016 / j.bbabio.2010.10.018), and it is secreted by Galdieria spp. as a response to O2 limitation (Sarian et aL, 2016, 'Effects of oxygen limitation on the biosynthesis of photo pigments in the red microalgae Galdieria sulphuraria strain 074G', PLOS One, 11 (2), doi: 10.1371 / journal.pone.0148358; Zhu et al., 2022, 'The thermoacidophilic red alga Galdieria sulphuraria is a highly efficient cell factory for ammonium recovery from ultrahigh-NH4+ industrial effluent with co-production of high-protein biomass by photo-fermentation.' Chemical Engineering Journal, 438, 135598, doi: 10.1016 / j.cej.2O22.135598) which leads to pigment degradation (chlorosis).

[0299] Net CO2 consumption rates were ~80 % slower in the cocultures that developed yellow coloration compared to the corresponding non-CH4 cocultures of equivalent mass ratio (p-value < 0.0001; Figure 5a). While this can be partially explained by the production of CO2 via CH4 oxidation (Figure 5b), the trend can also be seen for net C fixation (44 - 62% slower net C fixation rate, p-value < 0.001; Figure 5c). Therefore, in CH4-containing cocultures with an initial mass ratio > 0.23 (Methylacidiphilum Galdieria), Galdieria consumed less CO2 (Figure 5a) and produced less biomass (Figure 4b).

[0300] Increasing Methylacidiphilum starting concentrations coincided with increased net CH4 oxidation rates to a maximum of 0.124 ± 0.001 mmolcH4 L’1h-1at a Methylacidiphilum:Galdieria ratio of 0.15 (1.4-fold increase, p-value < 0.05; Figure 5b). At this ratio, CH4 was completely removed. Further increasing the Methylacidiphilum concentration in cocultures tended to diminish the amount of CH4 removed.

[0301] The decrease in CFU consumption with increasing Methylacidiphilum concentration is likely a consequence of limited O2 availability, as the net O2 production was 56-99 % lower (p-values < 0.0001) for CFU-containing cocultures with initial Methylacidiphilum:Galdieria ratios > 0.15, and approached 0 for ratios > 0.23 (Figure 5d).

[0302] At the highest ratios, some CH4 oxidation was observed (0.062 ± 0.016 mmolcH4 L’1h-1). Galdieria therefore must have initially been capable of producing O2, however, this would have been quickly consumed by Methylacidiphilum, causing O2 limitation. As a result, ODeoo increased only within the first 24 h of cultivation for CH4-containing cocultures with initial ratios > 0.15. The subsequent appearance of the yellow colouration, 1-2 days post O2 limitation, is consistent with photopigment damage (chlorosis) and a cessation of oxygenesis by Galdieria.

[0303] This shows that initial Methylacidiphilurrr.Galdieria mass ratios < 0.15 allowed for greater carbon fixation, while ratios > 0.23 negatively impacted Galdieria and hence coculture growth, CO2 fixation and CH4 oxidation.

[0304] To further understand the role of O2 concentration in cocultures, a series of batch cocultures were performed in 1 L pressure bottles with different gas concentrations. For all cocultures (with and without CH4 supplementation), an initial Methylacidiphilum:Galdieria mass ratio of -1.33 was used.

[0305] In the cocultures where no CH4 was added, after a 2-day lag phase Galdieria sp. RTK37.1 growth was not significantly different (p-value > 0.05) than previously observed axenic Galdieria. However, in the CH4-containing cocultures, no Methylacidiphilum sp. RTK17.1 growth was observed (ODeoo values showed no significant change, p-value > 0.05) and Galdieria sp. RTK37.1 ODeoo increased slightly on the first day of culture and then stagnated.

[0306] Collectively, these results suggest that, aside from a modest shading effect, Methylacidiphilum sp. RTK17.1 does not affect the photoautotrophic growth of Galdieria sp. RTK37.1, unless their relative concentration are sufficient to induce CH4-oxidation dependent O2 limitation.

[0307] This was confirmed in a series of cocultures where CH4, CO2 and O2 were replenished daily. In these cocultures, the total final ODeoo reached 3.5 A.U. within 4 days incubation. Despite Methylacidiphilum being much more concentrated than in the cocultures without CH4-addition, there was no significant difference in Galdieria growth rates (p-value > 0.05). Chlorophyll and phycocyanin accumulated throughout cultivation, and nocoproporphyrin was observed. Thus, in the presence of excess O2, the growth of Methylacidiphilum sp. RTK17.1 does not inhibit Galdieria.5. Example 5 — Inlet oxygen concentrationMaterials and methods

[0308] Steady state cultures of Methylacidiphilum sp. RTK17.1 (R1), Galdieria sp. RTK37.1 (R3), and a coculture of both microorganisms (R2) were established using three identical 1 L bioreactors (BioFlo 110; New Brunswick Scientific) with a 900 ml working volume using V4 media at pH 2.5 as described in Example 1. Temperature was maintained at 45°C, with agitation at 400 rpm for Galdieria and 250 rpm for Methylacidiphilum and the coculture. Filter-sterilised gas mixtures were supplied at 16 mL min-1. Inlet gas composition for Methylacidiphilum sp. RTK17.1 and the coculture was approximately 2.1% O2, 1.1% CH4, 74% CO2, balance N2 (all v / v); and for Galdieria sp. RTK37.1 was 3.0% O2, 64% CO2, balance N2 (all v / v). Light was supplied to the Galdieria sp. RTK37.1 and coculture reactors at 100 molPhotons m-2s’1measured at the centre of the empty reactor.

[0309] To initiate axenic cultures, reactors were inoculated to ODeoo 0.1 A.U. with the corresponding microorganism and grown in batch mode until an ODeoo of 1.0 A.U. was reached, after which the inlet and effluent pumps were activated. For the coculture, the reactor was inoculated to 0.1 A.U. ODeoo of Galdieria sp. RTK37.1, grown in batch until an ODeoo of 1.0 A.U. was reached, and then the pumps were started. After achieving steady state, < 1 mL Methylacidiphilum sp. RTK17.1 (ODeoo 1.20 A.U.) was added to the reactor, and a new steady state was achieved. Bioreactor working volume was maintained at 900 mL by automatic regulation of the culture level with a scavenging / effluent pump. For the axenic methanotroph and the coculture, media was supplied, and spent broth withdrawn at a constant flow rate of 150 mL day-1(D = 0.167 day-1). For the microalgal culture, a flowrate of 250 mL day-1was used (D = 0.278 day-1). For all reactors, 3 mL liquid and 40 mL inlet and outlet gas samples were harvested daily using aseptic technique for biomass (ODeoo) and gas measurements. Systems were considered 'steady state' when there was less than a 5% ODeoo variation over a three-day period.

[0310] To study the effect that O2 concentration has on steady state cultures and cocultures of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1, inlet feed gas O2 concentration was varied between 1% and 3% (v / v). For these experiments, the liquid flowrate and agitation rate for all chemostat reactors was set to 250 mL day-1(D = 0.278 day-1) and 400 rpm. Inlet feed gas flowrate was 16 L min-1for all reactors. Custom gas mixtures with the desired O2 concentration were prepared in compressed gas cylinders and supplied to the reactors. In total, four different inlet O2 concentrations were used: 1.0, 1.6, 2.1, and 3.0% (v / v). The feed gas concentrations for all experimental conditions are shown in Table 7.able 7. Inlet feed gas concentrations.ll gas concentrations shown are % v / v with the balance N2. Concentrations are triplicate measurements ± standard deviation.

[0311] The remaining experimental conditions were as described previously. For all reactors, 2 mL liquid and 40 mL inlet and outlet gas samples were collected daily using aseptic technique for biomass (ODeoo), pigment fluorescence, absorption spectra, and gas measurements. For each of the inlet O2 experimental conditions, after achieving steady state, 200 mL of outlet broth was collected to determine biomass concentration gravimetrically. Also, to determine protein content and amino acid profiles, biomass was collected daily and stored at 4°C. After 1 L of broth had accumulated, it was centrifuged at 12,000 rpm for 15 minutes. Supernatants were then discarded, and the resulting biomass pellets were stored at -20°C until needed.

[0312] A differential sedimentation, optical density and autofluorescence (DSOF) method was used to differentiate the contribution of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 to the coculture ODeoo. A 0.75 mL sample of coculture was harvested and the optical density, [Coculture]i, was measured using an Ultrospec 10 cell density meter (Amersham Bioscience, United Kingdom). If sample ODeoo was greater than 0.6, it was diluted and remeasured. Next, 200 pL coculture were transferred into a black, flat bottom (chimney well) 96-well microplate (Greiner Bio-One, Austria), and the fluorescence emission, F1, at 670 nm (590 nm excitation), was measured using a microplate reader (Varioskan Lux, Thermo Scientific). Following this, a 1.5 mL coculture sample was transferred into a 1.5 mL centrifuge tube and centrifuged at 380 x g for 20 seconds in a microcentrifuge. The resulting supernatant (0.75 mL) was then collected, and the ODeoo, [Supernatant]^ and fluorescence (F2) were measured as described for the coculture above. Finally, the ODeoo values for Galdieria sp. RTK37.1 and Methylacidiphilum sp. RTK17.1 in coculture were then calculated using Equations 1 and 2 respectively.

[0313] Equation 1 :

[0316] where:[Galdieria]? F? y = - = —[Galdieria]! FResults

[0317] To investigate the impact of O2 concentration on coculture interactions, inlet feed gas O2 concentration was varied between 1% and 3% (v / v) in parallel axenic andcoculture steady state bioreactors. For the Methylacidiphilum sp. RTK17.1 culture and the coculture, increasing feed gas O2 concentration from 1.0 to 2.1 % (v / v) increased the steady state biomass concentration 83 % and 72 % respectively (p-values < 0.001, Figure 6). Further incremental addition to inlet O2 concentration had no significant effect on the steady state biomass concentration in either case. For the coculture at 3.0 % O2, a maximal steady state biomass concentration of 0.263 ± 0.02 gow L’1was achieved. The Galdieria sp. RTK37.1 culture was not significantly affected by inlet O2 concentration at the concentrations tested.

[0318] Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 concentrations in the coculture chemostat (R2) were quantified using the DSOF method (Figure 7). A 2-way ANOVA was used to compare Methylacidiphilum sp. RTK17.1 concentration within axenic (R1) and coculture (R2) chemostats. Both the O2 inlet concentration and Galdieria presence had a significant effect (p-value < 0.0001) on the steady state biomass concentration of Methylacidiphilum sp. RTK17.1, with 69.75% of the observed variation attributed to inlet O2 concentration and 18.85% due to Galdieria presence / absence.

[0319] Axenic growth of Methylacidiphilum sp. RTK17.1 was limited by O2 at inlet concentrations between 1.0-2.1%, and the concentration of Methylacidiphilum sp. RTK17.1 in coculture was significantly greater than in axenic cultures for all nominal inlet O2 concentrations (Figure 7a).

[0320] In coculture, Methylacidiphilum concentrations only significantly increased when the O2 inlet was increased from 1.0 to 1.6 % (Figure 7a), which suggests that at inlet O2 > 1.6% Galdieria was able to provide sufficient O2 for the methanotroph to overcome this limitation. This was further evidenced by the coculture O2 consumption rates, which were constant between 1.6 and 3.0 % O2 inlet concentration.

[0321] A 2-way ANOVA was used to compare steady state Galdieria sp. RTK37.1 biomass concentrations within axenic and coculture chemostats. A significant effect (p-value< 0.0001) was found for O2 inlet concentration (43.16% variation), Methylacidiphilum presence (12.44% variation), and the interaction between both factors (41.93% variation).

[0322] Galdieria sp. RTK37.1 was present at a considerably lower concentration in coculture than axenic chemostat for O2 inlet concentrations < 1.6% (Figure 7b). At these O2 inlet concentrations, chlorophyll and phycocyanin contents were similarly lower, and coproporphyrin was detected (Figure 8).

[0323] Steady state dissolved oxygen values were significantly different between reactors (p-value < 0.001; Figure 9a) and changed with increasing inlet O2 concentrations (p- value < 0.0001). There was less dissolved O2 in the coculture than in the axenicMethylacidiphilum p. RTK17.1 culture for all inlet O2 concentrations, despite Galdieria supplementing O2 via photosynthetic activity and the coculture consuming significantly less O2 per gram dry weight of biomass (p-value < 0.001; Figure 9b). Rather, the additional photosynthetically produced O2 supported faster Methylacidiphilum growth, which consequently increased steady state methanotroph biomass concentrations (Table 8), leading to a reduction in dissolved O2 in the media.

[0324] In the axenic Galdieria sp. RTK37.1 culture, dissolved oxygen increased linearly with inlet O2 concentration (Figure 9a), whereas for both the axenic Methylacidiphilum culture and the coculture, there was little difference in D.O. at inlet O2 < 2.1 % (Figure 9a). At these concentrations, both systems were O2 limited and increasing O2 supply resulted in enhanced biomass production. In contrast, at O2 inlet concentrations > 2 % cultures were not O2 limited and dissolved oxygen increased linearly, as was observed in the Galdieria culture.

[0325] Galdieria's photosynthetic activity supported greater Methylacidiphilum biomass in coculture (Table 8). While O2 inlet concentration affected CH4 consumption rates in both axenic Methylacidiphilum culture and coculture (p-value < 0.001; Figure 9d), there was no significant difference in CH4 consumption rates between axenic Methylacidiphilum culture and coculture (p-value > 0.05). The presence of Galdieria allowed the Methylacidiphilum in coculture to produce a greater biomass yield on an equivalent amount of CH4 as the axenic methanotroph (p-value < 0.0001; Table 8) which is reflected in the coculture showing a slower specific CH4 consumption rate (Figure 9e).

[0326] The total biomass yield of the coculture was greater than either axenic culture (Table 8). This increase in biomass yield did not affect protein quality, as the obtained indispensable amino acid contents were similar to the values reported in Example 3 (data not shown). Inlet O2 concentration did not greatly affect the amino acid distribution for any cultures.Table 8. Biomass yields on methane for axenic Methylacidiphilum and coculture.Calculated for Methylacidiphilum RTK17.1 biomass only, grown in coculture with Galdieria sp. RTK37.1.Calculated for combined biomass of Methylacidiphilum RTK17.1 and Galdieria sp. RTK37.1 grown in coculture.6. Example 6 — High density fed-batch cocultureMaterials and methods

[0327] A coculture chemostat was switched to 2.1% inlet O2 feed gas and allowed to reach steady state. All other experimental conditions were set as described in Example 5. Once steady state was reached, the inlet and effluent pumps were turned off to allow batch growth.

[0328] Liquid (2 mL) and inlet and outlet gas samples (40 mL) were collected daily using aseptic technique. Gas samples were analysed for gas concentrations. Liquid samples were analysed for total biomass concentration (ODeoo), proportional biomass concentrations (via DSOF as described in Example 5), and pigment content. In addition, 10 mL aliquots were routinely collected from the reactor (with V4 media replacement) to measure biomass concentration gravimetrically. To avoid nitrogen limitation, 10 mL of ammonium chloride (40 g L’1) were added when total ODeoo reached 3.0, 6.0 or 9.0 AU. To avoid chlorosis, gas flowrate and / or light intensity was increased if O2 outlet concentration decreased below 1.6%, coproporphyrin excretion was detected, or coculture growth stopped. Cultivation was terminated upon the onset of stationary phase.Results

[0329] For industrial production of biomass such as single cell protein (SCP), concentrated cultures are desirable, as these facilitate biomass recovery and decrease overall costs. To approximate a semi-industrial bioprocess, a static liquid fed-batch coculture was performed under a continuous gas feeding regime.

[0330] At day 1, the coculture chemostat was switched to batch mode with an inlet gas flowrate of 16 mL min-1, an inlet gas composition of 2.1% O2, 1.0% CH4, 75% CO2, balance nitrogen, and 400 rpm agitation. To avoid N or O2 limitation, ammonium chloride was added when coculture O.D. surpassed 3.0, 6.0 and 9.0 A.U. at days 11, 24, and 35 respectively, and light intensity was increased to 150 and 200 nmol photons m-2s’1when Galdieria OD stagnated at days 18 and 32 respectively.

[0331] The coculture took 42 days to reach stationary phase (Figure 10a), with a maximum biomass concentration of 2.73 gow L’1achieved on day 38 (Figure 10b). During the first 11 days of culture, Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1concentrations increased linearly at similar rates. As the culture progressed, growth rates for both microorganisms slowed, with Galdieria growth rate decreasing down more than Methylacidiphilum (70 % and 39 % decrease in respectively by day 11).

[0332] In highly concentrated cultures, cell shading may account for some level of decreased growth of phototrophs. For example, Example 4 showed a small but significant decrease in the overall growth rate of Galdieria RTK37.1 at higher proportional abundance of Methylacidiphilum sp. RTK17.1. However, light attenuation does not fully explain Methylacidiphilum's slowing growth rate in this case, as the growth rates for Methylacidiphilum and Galdieria would be expected to be proportional, as O2 would become more limiting as Galdieria growth slowed down.

[0333] In CH4-replete cultures, volumetric CH4 consumption rates are expected to increase proportionally to methanotroph growth to support the additional biomass. However, CH4 consumption rates were constant for the duration of the experiment (data not shown), which suggests a CH4 mass transfer limitation. Consequently, Methylacidiphilum biomass concentrations increased linearly, and the specific growth rate slowed.

[0334] Dissolved oxygen values did not decrease sufficiently to induce coproporphyrin excretion (data not shown). As methanotroph growth was constrained by CH4 limitation, it was unable to overwhelm Galdieria and thus net O2 consumption rates were stable (data not shown).

[0335] On day 32, Galdieria reached stationary phase and pigments started to degrade (data not shown). Increasing light intensity did not enhance phototroph growth rate. As both ammonium and O2 were available, and no coproporphyrin excretion was detected, it is possible that another nutrient was depleted from the media. An alternative explanation would be photoinhibition. Following day 35, coculture O2 consumption rates notably increased until day 42 when Methylacidiphilum reached stationary phase.

[0336] The maximum coculture biomass concentration achieved was 2.73 gow L’1.7. Example 7 — Sequential cultureMaterials and methods

[0337] Cultures of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 were maintained as described in Example 1.

[0338] Sequential bioreactors were set up in which an input gas was fed into a first bioreactor containing a first microorganism, and the output gas from the first bioreactor fed into a second bioreactor comprising a second microorganism. Methylacidiphilum sp. RTK17.1and Galdieria sp. RTK37.1 were used as the two microorganisms, and both orientations were tested i.e. with gas flow from Methylacidiphilum culture to Galdieria culture, and vice versa.

[0339] Cultures were grown using V4 medium in batch mode as described in Example 1. Cultures comprising 850 ml V4 medium were inoculated with 50 ml of liquid culture and incubated at 45°C with agitation at 400 rpm and gas flow of 10 ml / min air combined with 10 ml / min of mixed gas comprising 5% CH4, 20% CO2, balance N2, for a total gas flow of 20 ml / min. Galdieria cultures were illuminated with - 100 pmol photons-m’2-s’1.

[0340] The batch cultures were turned to chemostat cultures by continuous addition of V4 medium into each bioreactor and continuous broth removal from each bioreactor. Media flow and dilution rates are shown for each bioreactor in Tables 9 and 10.

[0341] Cultures were grown until stable, and then measurements were taken over four days and averaged.Results — Methylacidiphilum to Galdieria

[0342] For a sequential bioreactor setup in which input gas is fed first to Methylacidiphilum RTK 17.1 (R1 reactor), then to Galdieria RTK 37.1 (R3 reactor), and then to output, measurements averaged over four days are shown in Table 9.Table 9. Sequential culture parameters for Methylacidiphilum to Galdieria.Parameters Average SD SD %Gas in to R1CH4(%) 2.5 0.1 2.3CO2(%) 8.7 0.1 0.7O2(%) 10.1 0.4 4.1N2(%) 78.7 0.4 0.5R1 — Methylacidiphilum RTK 17.1 pH 2.2 0.1 4.0Dissolved oxygen (mg / L) 1.6 0.0 0.3Temperature (°C) 45.0 0.0 0.0Agitation (rpm) 600.0 0.0 0.0Reactor volume (ml) 982.5 187.9 19.1Media flow in (ml / h) 9.4 0.2 1.7Dilution rate (h1) 0.010 0.0 20.3OD60O 1.5 0.1 7.2Gas out of R1CH4(%) 0.4 0.1 16.5CO2(%) 10.4 0.5 4.3O2(%) 6.9 0.7 10.7N2(%) 82.3 0.3 0.4R3 — Galdieria RTK 37.1 pH 2.2 0.0 0.0Dissolved oxygen (mg / L) 1.7 0.0 1.1Temperature (°C) 45.0 0.0 0.0Agitation (rpm) 400.0 0.0 0.0Reactor volume (ml) 927.5 5.0 0.5Media flow in (ml / h) 4.6 0.1 2.1Dilution rate (h-1) 0.005 0.0 1.9OD60O 1.6 0.1 9.2Gas out of R3CH4(%) 0.4 0.1 16.5CO2(%) 9.8 0.6 5.7O2(%) 7.6 1.0 13.5N2(%) 82.3 0.4 0.5Gas flow (ml / h) 19.1Gas capture (R1 + R3)CH4(%) 86.8CO2(%) -7.6O2(%) 28.0N2(%) 0.0Results — Galdieria to Methylacidiphilum

[0343] For a sequential bioreactor setup in which input gas is fed first to Galdieria RTK 37.1 (R3 reactor), then to Methylacidiphilum RTK 17.1 (R1 reactor), and then to output, measurements averaged over four days are shown in Table 10. Table 10. Sequential culture parameters for Galdieria to Methylacidiphilum.Parameters Average SD SD %Gas in to R3CH4(%) 2.4 0.0 0.0CO2(%) 9.1 0.1 0.6O2(%) 10.3 0.1 0.6N2(%) 78.2 0.1 0.1R3 — Galdieria RTK 37.1 pH 2.2 0.0 0.3Dissolved oxygen (mg / L) 2.6 0.0 0.7Temperature (°C) 45.0 0.0 0.0Agitation (rpm) 400.0 0.0 0.0Reactor volume (ml) 917.5 5.0 0.5Media flow in (ml / h) 4.4 0.2 3.9Dilution rate (h-1) 0.005 0.0 4.3OD6QO 1.7 0.1 7.4Gas out of R3CH4(%) 2.4 0.0 0.0CO2(%) 8.8 0.2 2.3O2(%) 10.7 0.3 2.8N2(%) 78.3 0.2 0.2R1 — Methylacidiphilum RTK 17.1 pH 2.3 0.1 2.6Dissolved oxygen (mg / L) 1.7 0.0 0.5Temperature (°C) 45.0 0.0 0.0Agitation (rpm) 600.0 0.0 0.0Reactor volume (ml) 817.5 5.0 0.6Media flow in (ml / h) 9.6 0.2 1.8Dilution rate (h-1) 0.012 0.0 2.2OD60O 1.4 0.0 1.8Gas out of R1CH4(%) 0.3 0.0 0.0CO2(%) 10.4 0.4 4.2O2(%) 7.4 0.2 2.4N2(%) 82.0 0.2 0.3Gas flow (ml / h) 19.1Gas capture (R3 + R1)CH4(%) 88.1CO2 (%) -8.9O2(%) 31.7N2 (%) 0.08. Example 8 — Alternative phototrophMaterials and methods

[0344] A coculture experiment was set up as described in Example 2, with the following modifications:

[0345] Galdieria sulphuraria UTEX 2919 was grown in batch within 1 L Duran Pressure Plus Bottles equipped with bromobutyl rubber stopper to OD 3.85. The coculture with Methylacidiphilum sp. RTK17.1 and Galdieria sulphuraria UTEX 2919 was run as a batch (no media addition) and with continuous gas flow (10ml / min 5%CH4 / 20%CO2 / N2 + 10ml / min air). The resulting gas composition entering the bioreactor was 2.3% CH4, 8.0% CO2, 10.9% O2, 78.1% N2.Results

[0346] Galdieria sulphuraria UTEX 2919 was able to grow in a bioreactor in the presence of Methylacidiphilum sp. RTK17.1 as shown in Table 11.Table 11. Coculture of Methylacidiphilum sp. RTK17.1 and Galdieria sulphuraria UTEX 2919.Day OD600 OD pH DO %CH4%CO2%O2%N2LED lightRatio (mg / L) out out out out (pmolA B(B:A) photons-nr2,S'1)0 0.19 0.17 0.89 2.55 2.81 2.3 8.0 10.9 78.1 1001 0.27 0.30 1.11 2.45 2.55 2.0 9.3 9.8 78.8 1002 0.44 0.38 0.86 2.37 2.17 1.4 9.6 8.9 80.0 1005 1.20 0.85 0.71 2.25 1.85 0.8 10.3 7.6 81.1 1006 1.70 1.10 0.65 2.24 1.92 0.9 10.2 7.8 81.0 1507 2.50 1.50 0.60 2.24 1.88 0.8 10.0 7.9 80.5 1508 3.90 1.60 0.41 2.24 1.87 0.8 9.7 7.8 80.8 1509 4.80 2.60 0.54 2.24 1.91 1.1 10.3 8.1 80.4 200A = methanotroph; B = microalgae.

[0347] The OD ratio of Microalgae:Methanotroph dropped over time, as the coculture became more turbid. On days 5-8, the gas composition exiting the bioreactor stabilised in average at 0.82% CFU, 10.0% CO2, 7.77% O2, 80.85% N2. This corresponds to a gas utilisation of 65% CH4, -21% CO2 and 31% O2.9. Example 9 — Additional methanotrophic bacteria and phototrophs

[0348] The experiments of Examples 1-7 are repeated using different methanotrophic bacteria and / or different phototrophs as shown in Table 12. Each different combination of methanotrophic bacteria and phototroph is tested.Table 12. Additional methanotrophic bacteria and phototrophs.Genus SpeciesMethanotrophic bacteriaMethylacidiphilum Sp. RTK17.1Methylacidiphilum fumariolicumMethylacidiphilum infernorumMethylacidiphilum kamchatkenseMethylacidiphilum Sp. YelMethylacidiphilum Sp. PhiMethylacidithermus pantelleriaMethylacidithermus RTKMethylacidimicrobium cyclophantesMethylacidimicrobium fagopyrumMethylacidimicrobium tartarophylaxMethylacidimicrobium Sp. LP2APhototrophsGaldieria Sp. RTK37.1Galdieria daedalaGaldieria maximaGaldieria partitaGaldieria phlegreaGaldieria sulphurariaCyanidioschyzon RTKCyanidioschyzon merolaeCyan id iococcus yangmingshanesisCyanidium RTKCyanidium caldariumCyanidium daedalumCyanidium maximumCyanidium partitumCyanidium rumpens

[0349] The various different combinations of methanotrophic bacteria and phototrophs are useful in the methods and systems disclosed herein.INDUSTRIAL APPLICATION

[0350] The methods and systems described herein are useful for producing biomass from an input gas, such as a waste gas stream from an industrial process. The biomass produced may be useful for a variety of purposes, such as a high protein component of nutritional compositions. The methods and systems described herein are also useful for decreasing the methane and / or carbon dioxide content of an input gas, thereby decreasing greenhouse gas emissions.

Claims

1. INDICATIVE CLAIMS1. A method for producing biomass from an input gas, the method comprising: a) coculturing one or more strains of methanotrophic bacteria and one or more strains of phototroph in the presence of the input gas and light to produce an output gas and biomass, or b) culturing one or more strains of methanotrophic bacteria in the presence of the input gas to produce an intermediate gas and biomass, and culturing one or more strains of phototroph in the presence of the intermediate gas and light to produce an output gas and biomass, or c) culturing one or more strains of phototroph in the presence of the input gas and light to produce an intermediate gas and biomass, and culturing one or more strains of methanotrophic bacteria in the presence of the intermediate gas to produce an output gas and biomass; wherein the one or more strains of methanotrophic bacteria and the one or more strains of phototroph are thermophilic, acidophilic, or thermoacidophilic.

2. A method for decreasing the carbon containing gas content (for example, the methane and / or carbon dioxide content) of an input gas, the method comprising: a) coculturing one or more strains of methanotrophic bacteria and one or more strains of phototroph in the presence of the input gas and light to produce an output gas, or b) culturing one or more strains of methanotrophic bacteria in the presence of the input gas to produce an intermediate gas, and culturing one or more strains of phototroph in the presence of the intermediate gas and light to produce an output gas, or c) culturing one or more strains of phototroph in the presence of the input gas and light to produce an intermediate gas, and culturing one or more strains of methanotrophic bacteria in the presence of the intermediate gas to produce an output gas; wherein the one or more strains of methanotrophic bacteria and the one or more strains of phototroph are thermophilic, acidophilic, or thermoacidophilic; andwherein the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph remove at least a portion of the carbon containing gas from the input gas and / or the intermediate gas.

3. A system for producing biomass from an input gas, the system comprising one or more strains of methanotrophic bacteria and one or more strains of phototroph, wherein the system is adapted to receive the input gas and to: a) coculture the one or more strains of methanotrophic bacteria and the one or more strains of phototroph in the presence of the input gas and light to produce an output gas, or b) culture the one or more strains of methanotrophic bacteria in the presence of the input gas to produce an intermediate gas, and culture the one or more strains of phototroph in the presence of the intermediate gas and light to produce an output gas, or c) culture the one or more strains of phototroph in the presence of the input gas and light to produce an intermediate gas, and culture the one or more strains of methanotrophic bacteria in the presence of the intermediate gas to produce an output gas; wherein the one or more strains of methanotrophic bacteria and the one or more strains of phototroph are thermophilic, acidophilic, or thermoacidophilic; and wherein, when in use, at least one of the one or more strains of methanotrophic bacteria and / or at least one of the one or more strains of phototroph produce biomass.

4. A system for decreasing the carbon containing gas content (for example, the methane and / or carbon dioxide content) of an input gas, the system comprising one or more strains of methanotrophic bacteria and one or more strains of phototroph, wherein the system is adapted to receive the input gas and to: a) coculture the one or more strains of methanotrophic bacteria and the one or more strains of phototroph in the presence of the input gas and light to produce an output gas, or b) culture the one or more strains of methanotrophic bacteria in the presence of the input gas to produce an intermediate gas, and culture the one or morestrains of phototroph in the presence of the intermediate gas and light to produce an output gas, or c) culture the one or more strains of phototroph in the presence of the input gas and light to produce an intermediate gas, and culture the one or more strains of methanotrophic bacteria in the presence of the intermediate gas to produce an output gas; wherein the one or more strains of methanotrophic bacteria and the one or more strains of phototroph are thermophilic, acidophilic, or thermoacidophilic; and wherein, when in use, at least one of the one or more strains of methanotrophic bacteria and / or at least one of the one or more strains of phototroph remove at least a portion of the carbon containing gas from the input gas and / or the intermediate gas.

5. The method of claim 1 or 2, further comprising the step of recirculating at least a portion of the output gas such that the culturing one or more strains of methanotrophic bacteria, the culturing one or more strains of phototroph, and / or the coculturing, is in the presence of the input and recirculated gases.

6. The system of claim 3 or 4, wherein the system is adapted to recirculate at least a portion of the output gas, and wherein the system is adapted to culture and / or coculture the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph in the presence of the input and recirculated gases.

7. The method or system of any previous claim, wherein the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph are thermoacidophilic.

8. The method or system of any previous claim, wherein the one or more strains of methanotrophic bacteria comprise a strain from the phylum Verrucomicrobiota, preferably a species of Methylacidiphilum, more preferably Methylacidiphilum sp. RTK17.1, KCTC accession number KCTC 15819BP dated 19 February 2024, or a derivative thereof.

9. The method or system of any previous claim, wherein the one or more strains of phototroph comprise a strain of algae, preferably a strain of microalgae, more preferably a strain from the family Cyanidiophyceae, more preferably a species of Galdieria, or most preferably Galdieria sp. RTK37.1, KCTC accession number KCTC 15846BP dated 7 March 2024, or a derivative thereof.

10. The method or system of any previous claim, wherein: a) the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph are able to grow at, or have optimal growth at, a temperature of at least about 40°C, preferably at least about 45°C, more preferably from about 45°C to about 55°C; and / or b) culturing the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph comprises incubating at a temperature of at least about 40°C, preferably at least 45°C, more preferably from about 45°C to about 55°C.

11. The method or system of any previous claim, wherein: a) the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph are able to grow at, or have optimal growth at, a pH of less than about 4.0, preferably less than about 3.0, more preferably from about 1.8 to about 2.5; and / or b) culturing the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph comprises culturing in a medium having a pH of less than about 4.0, preferably less than about 3.0, more preferably having a pH of from about 1.8 to about 2.5.

12. The method or system of any previous claim, wherein the light comprises sunlight and / or light provided by one or more light-emitting devices.

13. The method or system of any previous claim, wherein the light is at an intensity of at least about 30 pmol photons m’2s’1, preferably at least about 100 pmol photons m’2s’1.

14. The method or system of any previous claim, wherein the culturing and / or coculturing takes place at a pressure above atmospheric pressure, preferably at least about 100 mbar above atmospheric pressure, more preferably at least about 500 mbar above atmospheric pressure.

15. The method or system of any previous claim, wherein the initial dry weight mass ratio of the one or more strains of methanotrophic bacteria to the one or more strains of phototroph is less than about 0.5, preferably less than about 0.3, more preferably between about 0.1 and about 0.3, most preferably about 0.15.

16. The method or system of any previous claim, wherein the culturing and / or coculturing occurs in a culture medium, preferably an aqueous culture medium.

17. The method or system of claim 16, wherein the culture medium has a pH of less than about 4.0, preferably less than about 3.0, more preferably from about 1.8 to about 2.5.

18. The method or system of claim 16 or 17, wherein the culture medium comprises at least about 0.2 mg / L of dissolved oxygen, preferably at least about 1 mg / L of dissolved oxygen.

19. The method or system of any previous claim, wherein the input gas is at a temperature of at least about 35°C, preferably at least about 50°C, more preferably at least about 70°C.

20. The method or system of any previous claim, wherein the input gas comprises a waste gas stream from one or more industrial processes, preferably from power generation (such as geothermal power generation), oil production, and / or natural gas production.

21. The method or system of any previous claim, wherein the input gas comprises gas produced by the microbial degradation of organic material.

22. The method or system of any previous claim, wherein the input gas comprises at least about 0.1% v / v methane, preferably at least about 1.0% v / v methane.

23. The method or system of any previous claim, wherein the input gas comprises at least about 1.0% v / v oxygen, preferably at least about 2.0% v / v oxygen, more preferably about 3.0% v / v oxygen.

24. The method or system of any previous claim, wherein the input gas comprises at least about 10% v / v carbon dioxide, preferably at least about 50% v / v carbon dioxide, more preferably about 73% v / v carbon dioxide.

25. The method or system of any previous claim, wherein the input gas comprises from about 1.0% to about 15% v / v methane, from about 1.0% to about 20% v / v oxygen, and / or from about 10% to about 98% v / v carbon dioxide.

26. The method or system of any previous claim, wherein the input gas, intermediate gas, and / or output gas is not an explosive mixture.

27. The method or system of any previous claim, wherein the output gas has a reduced content of carbon containing gas (for example, methane and / or carbon dioxide) compared to the input gas.

28. The method or system of claim 27, wherein the output gas has a carbon containing gas content (for example, methane and / or carbon dioxide content) that is less than about 50% of that of the input gas.

29. The method or system of claim 27, wherein the output gas contains less than about 10% v / v methane, preferably less than about 1% v / v, more preferably less than about 0.1% v / v.

30. The method or system of any previous claim, wherein the biomass comprises: a) one or more biopolymers, preferably one or more proteins, glycoproteins, polypeptides, polysaccharides, and / or polynucleotides, b) one or more amino acids, c) one or more nucleotides, and / or nucleosides, d) one or more carbohydrates, e) one or more lipids, f) one or more vitamins, g) one or more bioactive compounds, preferably one or more pigmentcontaining compounds, more preferably phycocyanin, or h) any combination of any two or more of (a) to (g).

31. The method or system of any previous claim, wherein the biomass comprises at least about 20% w / w protein by dry weight, preferably at least about 30%, more preferably at least about 40%, most preferably at least about 50% w / w protein by dry weight.

32. The method or system of any previous claim, wherein the biomass has an essential amino acid concentration of at least about 6 g / 100g dry weight, preferably at least about 10 g / 100g dry weight, more preferably at least about 18 g / 100g dry weight.

33. The method or system of any previous claim, wherein the biomass comprises: a) at least about 2.5 g lysine per 100 g protein, preferably at least about 4.5 g per 100 g protein; b) at least about 1.0 g methionine per 100 g protein, preferably at least about 1.6 g per 100 g protein; c) at least about 0.4 g cysteine per 100 g protein, preferably at least about 0.6 g per 100 g protein; or d) any combination of any two or more of (a) to (c).

34. The method or system of any previous claim, wherein the biomass comprises protein that, if consumed as the sole dietary protein source at 0.66 g protein kg-1day-1, would meet or exceed the required daily human intake for: a) isoleucine, b) leucine, c) lysine, d) methionine, e) phenylalanine, f) threonine, g) tryptophan, h) valine, or i) any combination of any two or more of (a) to (h).

35. The method of any previous claim, further comprising the step of recovering biomass from the one or more strains of methanotrophic bacteria and / or the one or more strains of phototroph.

36. Biomass produced by the method or using the system of any previous claim.

37. Biomass comprising, or produced by, a coculture of Methylacidiphilum and Galdieria, preferably Methylacidiphilum sp. RTK17.1 KCTC accession number KCTC 15819BP dated 19 February 2024, or a derivative thereof, and Galdieria sp. RTK37.1 KCTC accession number KCTC 15846BP dated 7 March 2024, or a derivative thereof.

38. A nutritional composition comprising the biomass of claim 36 or 37.

39. A fertiliser composition comprising the biomass of claim 36 or 37.

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