A method for co-utilization of methane and carbon dioxide and system thereof

Abstract

The present disclosure relates to simple and efficient method for capture and bioconversion of carbon dioxide or biological reduction of carbon dioxide to high value products. Particularly, the present disclosure relates to methods for co-utilization of methane and carbon dioxide using methanotrophic bacteria with or without an optional microbe. The disclosure also relates to corresponding recombinant microbes, method of preparation of recombinant microbes, and systems thereof.

Description

A METHOD FOR CO-UTILIZATION OF METHANE AND CARBON DIOXIDE ANDSYSTEM THEREOFTECHNICAL FIELD

[0001] The present disclosure pertains to the field of biotechnology or molecular biology, particularly towards microbial conversion of greenhouse gases into value added products. More particularly, the present disclosure relates to a method of utilization of gaseous substrates such as methane (CH4) and carbon dioxide (CO2) by methanotrophic bacteria. The disclosure also relates to corresponding recombinant microbes, methods of preparation of recombinant microbes, and systems thereof.BACKGROUND OF THE DISCLOSURE

[0002] Background description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is relevant to the presently claimed disclosure, or that any publication specifically or implicitly referenced is prior art to the presently claimed disclosure.

[0003] Emission of greenhouse gases has been increasing globally at an alarming rate. Global anthropogenic emissions of carbon dioxide (CO2) and methane (CH4) have hit -43.1 billion tons and 9390 mmtCChe, respectively. As a result, fears about catastrophic global warming and climate change have intensified. Efforts to mitigate carbon dioxide emissions have focused on various approaches, including carbon capture and storage (CCS) and carbon capture and utilization (CCU). Among these, CCU has gained much attention due to its potential to convert carbon dioxide into valuable products, contributing to a circular carbon economy. Hence, strategies to convert greenhouse gases into value-added products are particularly appealing owing to potential environmental and economic benefits. Further, because of their abundance and low cost, both carbon dioxide and methane have gained attention for industrial usage as alternate feedstocks. However, as these exist in gaseous form and are stable in nature, their bioconversion becomes quite challenging.

[0004] For carbon dioxide fixation, currently the methods either rely on light (photosynthesis) or hydrogen supply (reduction). Both sources are challenging for industrial scale-up. A keyparameter influencing carbon dioxide utilization is the photosynthetic conversion efficiency, representing the luminous energy transformed to chemical energy, which is at the base of the anabolic reactions leading to biomass production. The maximum theoretical efficiency of solar energy conversion into biomass is around 12%. Open ponds and closed photobioreactor designs have been implemented at industrial scales for carbon dioxide utilization using photosynthetic algae. However, both designs have their own limitations, such as the requirement of large surface area and being highly prone to contamination. On the other hand, organisms such as hydrogenotrophic methanogens and autotrophic acetogens do not depend on light and rely on hydrogen as a reductant. Providing hydrogen gas at commercial scales during fermentation is expensive, invokes safety concerns due to its highly flammable nature and presents solubility issues as the gas is poorly soluble in aqueous solvents.

[0005] Co-utilization of carbon dioxide and methane has not been achieved yet on an industrial scale. Further, another limitation in the field of carbon capture / fixation faces is the cost of carbon dioxide abatement, which changes depending on the source of carbon dioxide as well as the technology that is used for abatement. The numbers reported in the literature vary widely and range from $30 to $300 / t CO2 abated. The current goal, which is considered ambitious, is to bring the cost of CO2 removing technology to $100 / t CO2.

[0006] Despite their potential advantages, challenges persist in developing robust and scalable methods and systems for efficient carbon dioxide utilization with or without methane. These challenges include providing sufficient light intensity at high cell densities, proper agitation, removal of oxygen, nutrient supply, and harvesting in case of algal strains and providing safe and sustainable supply of hydrogen for hydrogenotrophic bacterial cultures.

[0007] The present disclosure tries to address said need.OBJECTS OF THE DISCLOSURE

[0008] It is an object of the present disclosure to provide a simple and efficient method for capture and bioconversion of carbon dioxide or biological reduction of carbon dioxide to highvalue products. It is also an object of the present disclosure to reduce the cost of carbon dioxide abatement by providing an efficient method of capture and / or recycling of carbon.

[0009] It is another object of the present disclosure to provide a method for co-utilization of methane and carbon dioxide to produce value-added products.

[0010] It is another object of the present disclosure to provide a method for biological production and utilization of hydrogen for in vivo carbon dioxide reduction.

[0011] It is yet another object of the present disclosure to provide a recombinant methanotrophic bacteria that is capable of bioconversion and fixation of carbon dioxide and methane into value-added products. More particularly, the objective is to develop a recombinant methanotrophic bacteria that is capable of co-utilization of methane and carbon dioxide.

[0012] Still another object of the present disclosure is to develop an integrated system for the utilization of methane and carbon dioxide.SUMMARY

[0013] The present disclosure provides method of utilizing (CH4) and carbon dioxide (CO2) using a methanotroph with or without the help of another microbe. The method of the present disclosure also involves biological utilization of hydrogen for in vivo carbon reduction. Particularly, the present disclosure provides a method of utilizing methane (CH4) and carbon dioxide (CO2) to produce value-added product, comprising: culturing a methanotrophic bacteria in presence of the methane (CH4), and subjecting the methanotrophic bacteria to stress to produce an organic acid and hydrogen (H2); and culturing the methanotrophic bacteria and optionally a second microbe in presence of carbon dioxide (CO2) and at least one of the hydrogen and the organic acid to produce the value-added product.

[0014] In some embodiments, the method of the present disclosure is a single stage method that involves using a methanotroph capable of utilizing both CH4 and CO2.

[0015] In some embodiments, the method comprises- culturing a methanotrophic bacteria in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and- culturing the methanotrophic bacteria in presence of carbon dioxide (CO2), and hydrogen to produce the value-added product.

[0016] In some embodiments, the method of the present disclosure is a two-stage method that involves two stages: In the first stage, a methanotroph utilizes CH4 in a first bioreactor and under stress, produces organic acids and H2. In the second stage, a second microbe (wild-type or genetically engineered) is grown in a second bioreactor with the input of organic acids and H2 (produced by the methanotroph in the first bioreactor) and fixes or reduces CO2, thereby producing biomass and value added products.

[0017] In some embodiments, the method comprises:- culturing a methanotrophic bacteria in a first bioreactor in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and- culturing the methanotrophic bacteria in the first bioreactor and a second microbe in a second bioreactor in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce the value-added product.

[0018] In some embodiments, the method of the present disclosure is a single-stage cocultivation method that involves co-cultivation of a methanotroph and another microbe in a single bioreactor for simultaneous utilization of CH4 and CO2.

[0019] In some embodiments, the method comprises:- culturing a methanotrophic bacteria in presence of the methane (CH4), and subjecting the methanotrophic bacteria to stress to produce an organic acid and hydrogen (H2); and- culturing the methanotrophic bacteria and a second microbe in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce the value-added product, wherein the methanotrophic bacteria and the second microbe are present in a first / single bioreactor.

[0020] In some embodiments of the present method, the method is carried out at a temperature of about 5°C to about 50°C, including all the values and ranges therebetween.

[0021] In some embodiments of the present method, the method is carried out at a pH of about 3 to 8, including all the values and ranges therebetween.

[0022] In some embodiments of the present method, the method is carried out in a bioreactor selected from a stirred tank reactor, an airlift reactor, a bubble column reactor, or a plug flow reactor.

[0023] In some embodiments of the present method, a stirred tank reactor is used where the mixing takes place by agitating the reactor at about 200 rpm to 1000 rpm.

[0024] In some embodiments of the present method, a bubble column reactor is used where mixing takes place supplying gases from the bottom of the reactor vessel at a flow rate ranging between 0.05 wm to 3 wm.

[0025] In some embodiments of the present method, an airlift reactor is used where draft tubes are provided to segregate the gas flow into upward and downcomer. Typically, the gases are supplied from the bottom of the vessel in between the two draft tubes. Other configurations of the draft tubes are possible to change the gas mixing pattern.

[0026] In some embodiments of the present method, a plug flow reactor is used where gases are introduced at one end of the tube and mixing takes place by liquid and gas flow along the length of the tube. Different configuration of tubular arrangements can be made to enhance mass transfer and mixing.

[0027] In some embodiments of the present method, the value-added products comprise biomass (rich in lipids, proteins, nucleic acids), organic acids, single cell protein, antibacterial and anticancer drugs, amino acids, vitamins, industrial chemicals, industrial enzymes, biofuel, proteins, peptides, sugars, carbohydrates, fats, fat derivatives (acids, alcohols, acyl CoA, etc.), oils, pigments, secondary metabolites (including carotenoids, terpenoids and others) or combinations thereof.

[0028] The present disclosure also provides a method of culturing a microbe.

[0029] The present disclosure also provides recombinant microbes, particularly, recombinant or genetically modified methanotrophs. The recombinant methanotrophs of the present disclosure are able to utilize carbon dioxide apart from methane under specific cultivation conditions.

[0030] In some embodiments, the recombinant methanotrophic bacteria comprises at least one gene modification selected from a group comprising: i. overexpressed homologous or heterologous bacteriohemerythrin gene; ii. overexpressed homologous or heterologous genes or pathways for NAD(P)H synthesis or recycling; iii. overexpressed genes encoding one or more enzymes selected from a group comprising carboxylation systems (including carboxylases and carbonic anhydrases), central carbon metabolism, redox balance, and regeneration systems; iv. targeting any other genes involved in the central carbon metabolism that are involved in CO fixation,; v. knocked out native and a heterologous formate dehydrogenase (FDH) genes; or combinations thereof.

[0031] The method of the present disclosure optionally employs a second microbe - either wildtype or a genetically modified microbe.

[0032] In some embodiments, the second microbe is a bacteria, preferably a mixotrophic bacteria or a hydrogenotrophic bacteria.

[0033] The present disclosure also provides a vector. In some embodiments, the present disclosure provides a single vector or a single co-expression vector. In some embodiments, the present disclosure provides multiple vectors.

[0034] The present disclosure also refers to a system for utilizing methane and carbon dioxide and a system for culturing a microbe.BRIEF DESCRIPTION OF SEQUENCES OF THE PRESENT DISCLOSURESEQ ID NO. 1 refers to the nucleotide sequence of ccfdh gene of Clostridium carboxidivorans [GenBank: HM590561.1 (ORF: Ccar_1225); UmProt: E2IQB0)]SEQ ID NO. 2 refers to the nucleotide sequence of fdhD gene of Clostridium carboxidivorans [GenBank: HM590562.1 (ORF: Ccar_2671); UmProt: E2IQB1]SEQ ID NO. 3 refers to the nucleotide sequence of fits gene of Methylosinus trichosporium [GenBank: ATQ68555.1 (ORF: CQW49 12195); UmProtKB: A0A2D2D0N3]SEQ ID NO. 4 refers to the nucleotide sequence of zwf gene of Methylococcus capsulatus [GenBank: AAU90715.1; Gene: zwf-1 (MCA0025); UmProtKB: Q60CQ1]SEQ ID NO. 5 refers to the nucleotide sequence of ppc gene of Methylosinus trichosporium [GenBank: ATQ67339.1 (ORF: CQW49 05095); UmProtKB: A0A2D2CXG6]SEQ ID NO. 6 refers to the nucleotide sequence of pgi gene of Methylosinus trichosporium [GenBank: ATQ68160.1 (ORF: CQW49 09940); UmProtKB: A0A2D2CZJ7]SEQ ID NO. 7 refers to the nucleotide sequence of bhr gene of Methylococcus capsulatus [GenBank: AE017282.2 (ORF: MCA0715); UmProtKB: Q60AX2]SEQ ID NO. 8 refers to the nucleotide sequence of hoxF gene of Cupriavidus necatar [UmProtKB: P22317, NCBI Reference Sequence: WP_011154010.1]SEQ ID NO. 9 refers to the nucleotide sequence of hoxU gene of Cupriavidus necatar [UmProtKB: P22318, NCBI Reference Sequence: WP_011154011.1]SEQ ID NO. 10 refers to the nucleotide sequence of hoxY gene of Cupriavidus necatar [UmProtKB: P22319, NCBI Reference Sequence: WP_011154012.1]SEQ ID NO. 11 refers to the nucleotide sequence of hoxH gene of Cupriavidus necatar [UmProtKB: P22320, NCBI Reference Sequence: WP_011154013.1, GenBank: AY305378.1]SEQ ID NO. 12 refers to the nucleotide sequence of hoxW gene of Cupriavidus necatar [UmProtKB: Q79IP0, NCBI Reference Sequence: WP_011154014.1]SEQ ID NO. 13 refers to the nucleotide sequence of hoxl gene of Cupriavidus necatar [UmProtKB: Q79IN9, NCBI Reference Sequence: WP_011154015.1]SEQ ID NO. 14 refers to the nucleotide sequence of promoter pmxaFSEQ ID NO. 15 refers to the nucleotide sequence of promoter hpsSEQ ID NO. 16 refers to the nucleotide sequence of promoter fae2SEQ ID NO. 17 refers to the nucleotide sequence of promoter G54SEQ ID NO. 18 refers to the nucleotide sequence of promoter c70BREIT DESCRIPTION OF DRAWINGSFigure 1 is a flowchart that illustrates one embodiment of the method of the present disclosure.Figure 2 is a flowchart that illustrates an alternate embodiment of the method of the present disclosure.Figure 3 is a flowchart that illustrates yet another embodiment of the method of the present disclosure.Figure 4 is graph that illustrates the growth response of Methylosinus trichosporium under copper- limited (nutrient stress) conditions in a 5 L bioreactor system. It compares two cultivation setups — one supplied with methane (CH4) alone (thin line - X) and the other supplemented with both methane and carbon dioxide (CH4 + CO2; filled circle).Figure 5 illustrates graphs showing the secretion profiles of organic acids (succinic acid and acetic acid) by Methylosinus trichosporium under copper-limited (nutrient stress) conditions, with and without CO2 supplementation.Figure 6 illustrates graph demonstrating the growth behavior of Methylosinus trichosporium and Cupriavidus necator when co-cultured in a medium containing methane (CH4) and bicarbonate under different inoculation ratios.DESCRIPTION OF THE DISCLOSURE

[0035] The following is a detailed description of embodiments of the present disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0036] Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability.

[0037] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises”, “comprising”, “includes” and “including” are to be construed in an open, inclusive sense that is as “including, but not limited to.”

[0038] Reference throughout this specification to “one embodiment” or “some embodiments” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in some embodiments” in various places throughout this specification are notnecessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0039] As used in the description herein and throughout the numbered embodiments or claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0040] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, ratio and so forth, used to describe and claim certain embodiments of the disclosure are to be understood as being modified in some instances by the term “about”. Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

[0041] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0042] The headings and abstract of the disclosure provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0043] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein isintended merely to better illustrate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed.

[0044] The following discussion provides many exemplary embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0045] Various terms as used herein are described below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0046] The term “about” as used herein encompasses variations of + / -10% and more preferably -+7-5%, or any such variations that are appropriate for practicing the present disclosure to achieve desired effects.

[0047] As used herein, the term ‘methanotrophic’ organisms or ‘methanotrophs’ refers to prokaryotic microorganisms that can grow aerobically or anaerobically and metabolize singlecarbon compounds (such as methane) as their source of carbon and chemical energy for growth / survival. Similarly, the term ‘methanotrophic bacteria’ or ‘methanotroph bacteria’ refers to bacteria that can grow aerobically or anaerobically and metabolize single-carbon compounds (such as methane) as their source of carbon and chemical energy for growth / survival.

[0048] As used herein, the term “genetic engineering”, “genetic manipulation”, “recombination”, “recombinant DNA technology” and the likes are used interchangeably and refers to the act of modifying the genetic makeup / DNA of an organism. This modification for example, by way of introduction of a foreign gene / DNA into the organism, or manipulation of existing gene / DNA of the organism, to arrive at a recombinant organism. Thus, the term“recombinant” or the likes and variant terminologies thereof is also within the purview of the above definition. In some embodiments, the present disclosure provides a recombinant methanotrophic bacterium.

[0049] The term “recombinant” has been used interchangeably with “genetically modified” or “genetically engineered” throughout the specification.

[0050] As used herein, the term “carbon dioxide (CO2)” refers to carbon dioxide originating from any source, including but not limited to atmospheric CO2 naturally present in the culture environment, CO2 externally introduced into the system, and CO2 generated or evolved in situ as a metabolic by-product during methane (CH4) assimilation or oxidation by the microbial culture.

[0051] To overcome the drawbacks of the prior art and to meet the aforesaid objectives, the present disclosure provides a method of utilizing (CH4) and carbon dioxide (CO2) using a methanotroph with or without the help of another microbe. The method of the present disclosure also involves biological utilization of hydrogen for in vivo carbon reduction. It was surprisingly found that methanotrophic bacteria, which are known to utilize methane (CH4) as their primary carbon and energy source, are also capable of assimilating carbon dioxide (CO2) under specified conditions. This unexpected finding enables the concurrent / simultaneous utilization and recycling of two major greenhouse gases, i.e., methane and carbon dioxide, within a single biological system. The process not only reduces greenhouse gas emissions but also leads to the formation of value- added products such as organic acids. This, i.e., CH4 oxidation and CO2 fixation represents a non- obvious and an advantageous route for sustainable carbon conversion.

[0052] Particularly, the present disclosure provides a method of utilizing methane (CH4) and carbon di-oxide (CO2) to produce value-added products, comprising: culturing a methanotrophic bacteria in presence of the methane (CH4), and subjecting the methanotrophic bacteria to stress to produce an organic acid and hydrogen (H2); andculturing the methanotrophic bacteria and optionally a second microbe in presence of carbon dioxide (CO2), and at least one of the hydrogen (H2) and the organic acid to produce the value-added product.Methods of utiliz.ins methane and carbon dioxideMore particularly, the present disclosure provides methods of biological carbon reduction described in the following paragraphs.

[0053] In some embodiments, the method of the present disclosure is a single stage method that involves using a methanotroph capable of utilizing both CH4 and CO2 either simultaneously or in a stepwise manner, i.e., first step resulting in biomass growth with CH4 utilization and second step involving the utilization of CO2. The method in accordance with this embodiment of the present disclosure is illustrated in Figure 1.

[0054] In some embodiments, the method comprises- culturing a methanotrophic bacteria in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and- culturing the methanotrophic bacteria in presence of carbon dioxide (CO2), and hydrogen to produce the value-added product.

[0055] In some embodiments, the method of the present disclosure is a two-stage method that involves two stages: In the first stage, a wild-type or recombinant methanotroph utilizes methane (CH4) in the first bioreactor and under stress, produces organic acids and hydrogen (H2). In the second stage, a second microbe (wild-type or recombinant) is grown in a second / separate bioreactor with the input of organic acids and H2 (produced by the methanotroph in the first bioreactor) and fixes / reduces CO2 thereby resulting in the production of value added product. As per the need, H2 is supplied externally to the second bioreactor. The method in accordance with this embodiment of the present disclosure is illustrated in Figure 2.

[0056] In some embodiments, the method comprises:- culturing a methanotrophic bacteria in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and- culturing the methanotrophic bacteria and a second microbe in presence of carbon dioxide (CO2), the hydrogen, and the organic acid to produce the value-added product.

[0057] In some embodiments, the method comprises:- culturing a methanotrophic bacteria in a first bioreactor in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and- culturing the methanotrophic bacteria in the first bioreactor and a second microbe in a second bioreactor in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce the value-added product.

[0058] In some embodiments, the method comprises:- culturing a methanotrophic bacteria in a first bioreactor in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and- culturing the methanotrophic bacteria in the first bioreactor and a second microbe in a second bioreactor in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce the value-added product; wherein the organic acid and hydrogen from the first bioreactor is introduced into the second bioreactor and utilized by the second microbe to produce the value-added product.

[0059] In some embodiments, the method comprises: i. culturing a wild-type or recombinant methanotrophic bacteria in a first bioreactor in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2);wherein the methanotrophic bacteria uses the hydrogen in presence of CO2 to produce the value-added products; ii. introducing the organic acid and / or hydrogen (H2) produced in step i) into a second bioreactor having a wild-type or recombinant second microbe; wherein the second microbe utilizes the organic acid and / or hydrogen in presence of CO2 to produce value-added products.

[0060] In some embodiments, the method of the present disclosure is a single-stage cocultivation method that involves co-cultivation of a methanotroph and a second microbe in a single bioreactor for simultaneous utilization of CH4 and CO2. Although methanotroph primarily utilizes CH4, both organisms contribute towards fixation of CO2 simultaneously. The method in accordance with this embodiment of the present disclosure is illustrated in Figure 3.

[0061] In some embodiments, the method involves co-culture of a wild-type or recombinant methanotroph and different mixotrophs. Methanotrophs under stress (such as nutrient stress or microaerobic condition) produce organic acids and hydrogen. Co-cultured carbon dioxide-utilizing mixotrophs then utilize organic acids and hydrogen produced by the methanotroph to fix carbon dioxide thereby producing value added product. The mixotrophic strain either evolved and selected by applying selection criteria or produced by genetic engineering approaches.

[0062] In some embodiments, the method comprises:- culturing a methanotrophic bacteria in presence of the methane (CH4), and subjecting the methanotrophic bacteria to stress to produce an organic acid and hydrogen (H2); and- culturing the methanotrophic bacteria and a second microbe in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce the value-added product, wherein the methanotrophic bacteria and the second microbe are present in a first / single bioreactor.

[0063] In some embodiments, the method comprises:- culturing a methanotrophic bacteria in presence of the methane (CH4), and subjecting the methanotrophic bacteria to stress to produce an organic acid and hydrogen (H2); and- culturing the methanotrophic bacteria and a second microbe in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce the value-added product, wherein the methanotrophic bacteria and the second microbe are present in a first / single bioreactor. wherein the methanotrophic bacteria utilizes the hydrogen in presence of CO2 to produce the value-added products and the second microbe utilizes the hydrogen or the organic acids or both, in presence of CO2 to produce the value-added products.

[0064] In some embodiments, the methanotrophic bacteria is a wild type methanotrophic bacteria or a recombinant methanotrophic bacteria.

[0065] In some embodiments, the wild-type or recombinant methanotrophic bacteria is selected from a type I, type II, type X, or a Verrumicrobial methanotroph.

[0066] In some embodiments, the wild-type or recombinant methanotrophic bacteria is type I or type II or type X methanotroph belonging to genus selected from a group comprising Methylosinus spp. Methylococcus spp., Methylomonas spp., Methylomicrobium spp., Halomonas spp., Methylosarcina spp., Methylocaldum spp., Methylogaea spp., Methylosoma spp., Methyloparacoccus spp., Methyloglobulus spp., Methyloprofundus spp., Methylomarinum spp., Methylovulum spp., Methylomagnum spp., Methylosphaera spp., Methylohalobius spp., Methylothermus spp., Methylomarinovum spp., Acidithiobacillus spp., Crenothrix spp., Clonothrix spp., Methylocystis spp., Methylocucumis spp., Methyloferula spp.,., Methylocella spp., Methylocapsa spp., Methylacidiphilum spp., Methylacidimicrobium spp., Methylomirabilis spp., Methanoperedens spp., Mycolicibacterium spp., and combinations thereof

[0067] In some embodiments, the wild-type or recombinant methanotrophic bacteria is type I or type II or type X methanotroph belonging to genus selected from a group comprisingA7c / / 7j7 c cc / .s capsulatus, Methylococcus bovis, Methylococcus chroococcus, Methylococcus geothermalis, Methylococcus luteus, Methylococcus mesophilus, Methylococcusmobilis, Methylococcus thermophilus, Methylococcus vinelandii, Methylococcus whittenburyi, Methylomonas agile, Methylomonas albus, Methylomonas aurantiaca, Methylomonas aurea, Methylomonas defluvii, Methylomonas denitrificans, Methylomonas flagellata, Methylomonas fluvii, Methylomonas fodinarum, Methylomonas gracilis, Methylomonas koyamae, Methylomonas lenta, Methylomonas methanica, Methylomonas methanolica, Methylomonas methanooxidans, Methylomonas methylovora, Methylomonas montana, Methylomonas paludis, Methylomonas pelagica, Methylomonas rapida, Methylomonas rhizoryzae, Methylomonas rivi, Methylomonas rosea, Methylomonas rubra, Methylomonas scandinavica, Methylomonas subterranea, Methylomicrobium agile, Methylomicrobium album, Methylomicrobium alcaliphilum, Methylomicrobium buryatense, Methylomicrobium japanense, Methylomicrobium kenyense, Methylomicrobium lacus, Methylomicrobium pelagicum, Halomonas pantelleriensis, Methylosarcina fibrata, Methylosarcina lacus, Methylosarcina quisquiliarum, Methylocaldum gracile, Methylocaldum marinum, Methylocaldum szegediense, Methylocaldum tepidum, Methylogaea oryzae, Methylosoma difficile, Methyloparacoccus murrellii, Methyloglobulus morosus, Methyloprofundus sedimenti, Methylomarinum vadi, Methylovulum miyakonense, Methylovulum psychrotolerans, Methylomagnum ishizawai, Methylosphaera hansonii, Methylohalobius crimeensis, Methylothermus subterraneus, Methylothermus thermalis, Methylomarinovum caldicuralii, Methylomarinovum tepidoasis, Acidithiobacillus ferrivorans, Crenothrix polyspora, Clonothrix fusca, Methylocystis methanolicus, Methylocucumis oryzae, Methylogaea oryzae, Methylosarcina lacus, Methylosoma difficile, Methyloferula stellata, Methylosinus trichosporium, Methylocystis borborid, Methylocystis bryophila, Methylocystis echinoides, Methylocystis heyeri, Methylocystis hirsuta, Methylocystis iwaonis, Methylocystis minimus, Methylocystis parva, Methylocystis parvus, Methylocystis rosea, Methylocystis silviterrae, Methylocystis suffitae, Methylosinus pucelana, Methylosinus sporium, Methylocella palustris, Methylocella silvestris, Methylocella tundrae, Methylocapsa acidiphila, Methylocapsa aurea, Methylocapsa gorgona, Methylocapsa paisarum, Methyloferula stellata, Methylacidiphilum caldifontis, Methylacidiphilum jumariolicum, Methylacidiphilum infemorum, Methylacidiphilum kamchatkense, Methylacidimicrobium cyclopophantes, Methylacidimicrobium fagopyrum, Methylacidimicrobium tartarophylax, Methylacidimicrobium thermophilum, Methylomirabilis iodofontis, Methylomirabilis lanthanidiphila, Methylomirabilis limnetica, Methylomirabilis nitratireducens, Methylomirabilis oxyfera, Methylomirabilis oxygeniifera,Methylomirabilis sinica, Methylomirabilis tolerans, Methanoperedens ferrireducens, Methanoperedens manganicus, Methanoperedens manganireducens, Methanoperedens nitratireducens, Methanoperedens nitroreducens, Methanoperedens psychrophilus. and combinations thereof.

[0068] In some embodiments, the methanotroph is a wild type Methylosinus trichosporium.

[0069] In some embodiments, the methanotroph is a recombinant Methylosinus trichosporium.

[0070] In some embodiments, the methanotroph is Methylococcus capsulatus.

[0071] In some embodiments, the second microbe is a bacterium.

[0072] In some embodiments the second microbe is a wild type bacteria or a recombinant bacteria.

[0073] In some embodiments, the second microbe is a mixotrophic bacteria or a hydrogenotrophic bacteria.

[0074] In some embodiments, the second microbe is a bacteria selected from a group consisting of second microbe is selected from a group comprising Cupriavidus spp., Escherichia spp., Aquifex spp., Clostridium spp., Corynebacterium spp., Gordonia spp., Nocardia spp., Rhodobacter spp., Rhodopseudomonas spp., Rhodospirillum spp., Rhodococcus spp., Rhizobium spp., Thiocapsa spp., Pseudomonas spp., Hydrogenomonas spp., Hydrogenobacter spp., Hydrogenophilus spp., Hydrogenovibrio spp., Hydrogenothermus spp., Helicobacter spp., Xanthobacter spp., Hydrogenophaga spp., Bradyrhizobium spp., Ralstonia spp., Alcaligenes spp., Amycolata spp., Aquaspirillum spp., Arthrobacter spp., Azospirillum spp., Variovorax spp., Acidovorax spp., Bacillus spp., Calderobacterium spp., Derxia spp., Flavobacterium spp., Microcyclus spp., Mycobacterium spp., Paracoccus spp., Persephonella spp., Renobacter spp., Streptomyces spp., Thermocrinis spp., Wautersia spp., and combinations thereof.

[0075] In some embodiments, the second microbe is a bacteria selected from a group consisting of Cupriavidus necator, Escherichia coli, Aquifex pyrophilus, Aquifex aeolicus, Cupriavidus metallidurans, Clostridium autoethanogenum, Corynebacterium autotrophicum, Gordonia desulfuricans, Gordonia polyisoprenivorans, Gordonia rubripertincta, Gordonia hydrophobica,Gordonia westfalica, Nocardia autotrophica, Nocardia opaca, purple non-sulfiir photosynthetic bacteria including Rhodobacter sphaeroides, Rhodopseudomonas palustris, Rhodopseudomonas capsulata, Rhodopseudomonas viridis, Rhodopseudomonas sulfoviridis, Rhodopseudomonas blastica, Rhodopseudomonas spheroides, Rhodopseudomonas acidophila, Rhodospirillum rubrum, Rhodococcus opacus, Rhizobium japonicum, Thiocapsa roseopersicina, Pseudomonas facilis, Pseudomonas flava, Pseudomonas putida, Pseudomonas hydrogenovora, Pseudomonas hydrogenothermophila, Pseudomonas palleronii, Pseudomonas pseudoflava, Pseudomonas saccharophila, Pseudomonas thermophila, Hydrogenomonas pantotropha, Hydrogenomonas eutropha, Hydrogenomonas facilis, Hydrogenobacter thermophilus, Hydrogenobacter halophilus, Hydrogenobacter hydrogenophilus, Hydrogenophilus islandicus, Hydrogenovibrio marinus, Hydrogenothermus marinus, Helicobacter pylori, Xanthobacter autotrophicus, Xanthobacter flavus, Hydrogenophaga flava, Hydrogenophaga palleronii, Hydrogenophaga pseudoflava, Bradyrhizobium japonicum, Ralstonia eutropha, Alcaligenes eutrophus, Alcaligenes facilis, Alcaligenes hydrogenophilus, Alcaligenes latus, Alcaligenes paradoxus, Alcaligenes ruhlandii, Amycolata sp., Aquaspirillum autotrophicum, Arthrobacter strain 11 / X, Arthrobacter methylotrophus, Azospirillum lipoferum, Variovorax paradoxus, Acidovorax facilis, Bacillus schlegelii, Bacillus tusciae, Calderobacterium hydrogenophilum, Derxia gummosa, Flavobacterium autothermophilum, Microcyclus aquaticus, Mycobacterium gordoniae, Paracoccus denitrificans, Persephonella marina, Persephonella guaymasensis, Renobacter vacuolatum, Streptomycetes coelicoflavus, Streptomycetes griseus, Streptomycetes xanthochromogenes, Streptomycetes thermocarboxydus, Thermocrinis ruber, and Wautersia sp., as well as microbial consortia that include oxyhydrogen microorganisms.

[0076] In some embodiments, the second microbe exhibits the following traits:- efficient utilization of CO2- ability to utilize H2 and / or organic acids produced by the methanotroph, and- production of novel metabolites / target molecules.

[0077] In some embodiments of the present method, the stress is nutrient stress or microaerobic cultivation.

[0078] In some embodiments, the nutrient stress comprises limited oxygen (O2) supply, limited nitrogen (N) supply, limited phosphorus (P) supply, limited carbon (C) supply, limited copper (Cu) supply, limited iron (Fe) supply, limited sulfur (S) supply, limited magnesium (Mg) supply, limited potassium (K) supply, or combinations thereof.

[0079] In some embodiments of the single stage method, the stress is nutrient stress, preferably limited copper (Cu) supply.

[0080] In some embodiments of the present method, the method is carried out at a temperature of about 5°C to about 50°C, including all the values and ranges therebetween.

[0081] In some embodiments of the present method, the method is carried out at a temperature of about 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or 50°C, including all the values and ranges therebetween.

[0082] In some embodiments of the present method, the method is carried out at a pH of about 3 to 8, including all the values and ranges therebetween.

[0083] In some embodiments of the present method, the method is carried out at a pH of about 3, 4, 5, 6, 7, or 8, including all the values and ranges therebetween.

[0084] In some embodiments of the present method, the method is carried out in a bioreactor selected from a stirred tank reactor, an airlift reactor, a bubble column reactor, or a plug flow reactor.

[0085] In some embodiments of the present method, a stirred tank reactor is used where the mixing takes place by agitating the reactor at about 200 rpm to 1000 rpm.

[0086] In some embodiments of the present method, a bubble column reactor is used where mixing takes place supplying gases from the bottom of the reactor vessel at a flow rate ranging between 0.05 wm to 3 wm.

[0087] In some embodiments of the present method, an airlift reactor is used where draft tubes are provided to segregate the gas flow into upward and downcomer. Typically, the gases aresupplied from the bottom of the vessel in between the two draft tubes. Other configurations of the draft tubes are possible to change the gas mixing pattern.

[0088] In some embodiments of the present method, a plug flow reactor is used where gases are introduced at one end of the tube and mixing takes place by liquid and gas flow along the length of the tube. Different configuration of tubular arrangements can be made to enhance mass transfer and mixing.

[0089] In some embodiments of the present method, the culturing is carried out by batch fermentation, fed-batch fermentation, or continuous fermentation.

[0090] In some embodiments of the present method, the organic acid is selected from a group comprising acetic acid, lactic acid, citric acid, succinic acid, fumaric acid, or any intermediates of TCA cycle, or combinations thereof.

[0091] In some embodiments of the present method, the value-added product comprise biomass (rich in lipids, proteins, nucleic acids), organic acids, single cell protein, antibacterial and anticancer drugs, amino acids, vitamins, industrial chemicals, industrial enzymes, biofuel, proteins, peptides, sugars, carbohydrates, fats, fat derivatives (acids, alcohols, acyl CoA, etc.), oils, pigments, secondary metabolites (including carotenoids, terpenoids and others) or combinations thereof.

[0092] In some embodiments, the method comprises:- culturing a wild-type or recombinant methanotrophic bacteria in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and- culturing the methanotrophic bacteria in presence of carbon dioxide (CO2), and hydrogen to produce the value-added product; wherein the methanotrophic bacteria is selected from a group comprising Methylococcus spp., Methylomonas spp., Methylomicrobium spp., Halomonas spp., Methylosarcina spp., Methylocaldum spp., Methylogaea spp., Methylosoma spp., Methyloparacoccus spp., Methyloglobulus spp., Methyloprofundus spp., Methylomarinumspp., Methylovulum spp., Methylomagnum spp., Methylosphaera spp., Methylohalobius spp., Methylothermus spp., Methylomarinovum spp., Acidithiobacillus spp., Crenothrix spp., Clonothrix spp., Methylocystis spp., Methylocucumis spp., Methyloferula spp., Methylosinus spp., Methylocella spp., Methylocapsa spp., Methylacidiphilum spp., Methylacidimicrobium spp., Methylomirabilis spp., Methanoperedens spp., Mycolicibacterium spp. and combinations thereof.

[0093] In some embodiments, the method comprises:- culturing a wild-type or recombinant methanotrophic bacteria in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and- culturing the methanotrophic bacteria in presence of carbon dioxide (CO2), and hydrogen to produce the value-added product; wherein the methanotrophic bacteria is selected from a group comprising Methylococcus spp., Methylomonas spp., Methylomicrobium spp., Halomonas spp., Methylosarcina spp., Methylocaldum spp., Methylogaea spp., Methylosoma spp., Methyloparacoccus spp., Methyloglobulus spp., Methyloprofundus spp., Methylomarinum spp., Methylovulum spp., Methylomagnum spp., Methylosphaera spp., Methylohalobius spp., Methylothermus spp., Methylomarinovum spp., Acidithiobacillus spp., Crenothrix spp., Clonothrix spp., Methylocystis spp., Methylocucumis spp., Methyloferula spp., Methylosinus spp., Methylocella spp., Methylocapsa spp., Methylacidiphilum spp., Methylacidimicrobium spp., Methylomirabilis spp., Methanoperedens spp., Mycolicibacterium spp., and combinations thereof; wherein the method is carried out at a temperature of about 5°C to 50°C and a pH of about 3 to 8; and wherein the culturing is carried out by batch fermentation, fed-batch fermentation, or continuous fermentation.

[0094] In some embodiments, the method comprises:- culturing a wild-type or recombinant methanotrophic bacteria in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acid and hydrogen (H2); and- culturing the methanotrophic bacteria in presence of carbon dioxide (CO2), and hydrogen to produce the value-added product; wherein the methanotrophic bacteria is selected from a group comprising Methylococcus capsulatus Methylosinus trichosporium, Methylomicrobium alcaliphilum Methylomicrobium buryatense, Methylmicrobium kenyense or Methylohalobius crimeensis. wherein the method is carried out at a temperature of about 5°C to about 50°C at a pH of about 3 to 8; and wherein the culturing is carried out by batch fermentation.

[0095] In some embodiments, the method comprises:- culturing Methylosinus trichosporium bacteria in presence of methane (CH4), and subjecting the Methylosinus trichosporium bacteria to nutrient stress to produce organic acids and hydrogen (H2); and- culturing the methanotrophic bacteria in presence of carbon dioxide (CO2), and hydrogen to produce the value-added product; wherein the methanotrophic bacteria uses the hydrogen in presence of CO2 to produce the value-added products; wherein the nutrient stress is limited copper availability in the culture medium; wherein the method is carried out at a temperature of about 5°C to 50°C and a pH of about 3 to 8; and wherein the culturing is carried out by batch fermentation.

[0096] In some embodiments, the method comprises:- culturing Methylosinus trichosporium bacteria in presence of methane (CH4), and subjecting the Methylosinus trichosporium bacteria to nutrient stress to produce organic acids and hydrogen (H2); and- culturing the methanotrophic bacteria in presence of carbon dioxide (CO2), and hydrogen to produce the value-added product; wherein the methanotrophic bacteria uses the hydrogen in presence of CO2 to produce the value-added products; wherein the nutrient stress is limited copper availability; and wherein the organic acids are succinic acid and acetic acid.

[0097] In some embodiments, the method comprises:- culturing a methanotrophic bacteria in a first bioreactor in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and- culturing the methanotrophic bacteria in the first bioreactor and a second microbe in a second bioreactor in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce the value-added product; wherein the organic acid and the hydrogen from the first bioreactor is introduced into the second bioreactor and utilized by the second microbe to produce the value-added product; wherein the methanotrophic bacteria is selected from a group comprising Methylococcus spp., Methylomonas spp., Methylomicrobium spp., Halomonas spp., Methylosarcina spp., Methylocaldum spp., Methylogaea spp., Methylosoma spp., Methyloparacoccus spp., Methyloglobulus spp., Methyloprofundus spp., Methylomarinum spp., Methylovulum spp., Methylomagnum spp., Methylosphaera spp., Methylohalobius spp., Methylothermus spp., Methylomarinovum spp., Acidithiobacillus spp., Crenothrix spp., Clonothrix spp., Methylocystis spp., Methylocucumis spp., Methyloferula spp., Methylosinus spp., Methylocella spp., Methylocapsa spp., Methylacidiphilum spp.,Methylacidimicrobium spp., Methylomirabilis spp., Methanoperedens spp., Mycolicibacterium spp., and combinations thereof; and wherein the second microbe is a wild type or a recombinant mixotroph bacteria, or a hydrogenotrophic bacteria selected from a group comprising Cupriavidus spp., Escherichia spp., Aquifex spp., Clostridium spp., Corynebacterium spp., Gordonia spp., Nocardia spp., Rhodobacter spp., Rhodopseudomonas spp., Rhodospirillum spp., Rhodococcus spp., Rhizobium spp., Thiocapsa spp., Pseudomonas spp., Hydrogenomonas spp., Hydrogenobacter spp., Hydrogenophilus spp., Hydrogenovibrio spp., Hydrogenothermus spp., Helicobacter spp., Xanthobacter spp., Hydrogenophaga spp., Bradyrhizobium spp., Ralstonia spp., Alcaligenes spp., Amycolata spp., Aquaspirillum spp., Arthrobacter spp., Azospirillum spp., Variovorax spp., Acidovorax spp., Bacillus spp., Calderobacterium spp., Derxia spp., Flavobacterium spp., Microcyclus spp., Mycobacterium spp., Paracoccus spp., Persephonella spp., Renobacter spp., Streptomyces spp., Thermocrinis spp., Wautersia spp., and combinations thereof.

[0098] In some embodiments, the method comprises: i. culturing a wild-type or recombinant methanotrophic bacteria in a first bioreactor in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); wherein the methanotrophic bacteria uses the hydrogen in presence of CO2 to produce the value-added products; ii. introducing the organic acids and / or hydrogen (H2) produced in step i) into a second bioreactor having a wild-type or recombinant second microbe; wherein the second microbe utilizes the organic acid and / or hydrogen in presence of CO2 to produce value-added products; wherein the methanotrophic bacteria is selected from a group comprising Methylococcus spp., Methylomonas spp., Methylomicrobium spp., Halomonas spp., Methylosarcina spp., Methylocaldum spp., Methylogaea spp., Methylosoma spp.,Methyloparacoccus spp., Methyloglobulus spp., Methyloprofiindus spp., Methylomarinum spp., Methylovulum spp., Methylomagnum spp., Methylosphaera spp., Methylohalobius spp., Methylothermus spp., Methylomarinovum spp., Acidithiobacillus spp., Crenothrix spp., Clonothrix spp., Methylocystis spp., Methylocucumis spp., Methyloferula spp., Methylosinus spp., Methylocella spp., Methylocapsa spp., Methylacidiphilum spp., Methylacidimicrobium spp., Methylomirabilis spp., Methanoperedens spp., Mycolicibacterium spp., and combinations thereof; wherein the second microbe is a wild-type or a genetically modified mixotrophic bacteria; wherein the method is carried out at a temperature of about 5°C to about 50°C at a pH of about 3 to 8; and wherein the culturing is carried out by batch fermentation, fed-batch fermentation, or continuous fermentation.

[0099] In some embodiments , the method comprises: i. culturing a wild-type or recombinant methanotrophic bacteria in a first bioreactor in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); wherein the methanotrophic bacteria uses the hydrogen in presence of CO2 to produce the value-added products; ii. introducing the organic acids and / or hydrogen (H2) produced in step i) into a second bioreactor having a wild-type or recombinant second microbe; wherein the second microbe utilizes the organic acids and / or hydrogen in presence of CO2 to produce value-added products; wherein the methanotrophic bacteria is Methylosinus trichosporiunr, wherein the second microbe is Cupriavidus necator,wherein the culturing is carried out by batch fermentation at a temperature of about 5°Co about 50°C at a pH of about 3 to 8.

[0100] In some embodiments, the method comprises:- culturing Methylosinus trichosporium bacteria in a first bioreactor in presence of methane (CH4), and subjecting the Methylosinus trichosporium bacteria to nutrient stress, i.e., limited Cu availability to produce organic acids and hydrogen (H2); and- culturing the Methylosinus trichosporium in the first bioreactor and Cupriavidus necator in a second bioreactor in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce the value-added product; wherein the organic acid and hydrogen from the first bioreactor is introduced into the second bioreactor; and wherein culturing is carried out by batch fermentation at a temperature of about 5°C to about 50°C at a pH of about 3 to 8.

[0101] In some embodiments, the method comprises:- culturing a methanotrophic bacteria in presence of the methane (CH4), and subjecting the methanotrophic bacteria to stress to produce an organic acid and hydrogen (H2); and- culturing the methanotrophic bacteria and a second microbe in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce the value-added product, wherein the methanotrophic bacteria and the second microbe are present in a first / single bioreactor; wherein the methanotrophic bacteria utilizes the hydrogen in presence of CO2 to produce the value-added products and the second microbe utilizes the hydrogen or the organic acids or both, in presence of CO2 to produce the value-added products; wherein the methanotrophic bacteria is selected from a group comprising Methylococcus spp., Methylomonas spp., Methylomicrobium spp., Halomonas spp., Methylosarcina spp., Methylocaldum spp., Methylogaea spp., Methylosoma spp., Methyloparacoccus spp., Methyloglobulus spp., Methyloprofundus spp., Methylomarinum spp., Methylovulum spp., Methylomagnum spp., Methylosphaera spp., Methylohalobiusspp., Methylothermus spp., Methylomarinovum spp., Acidithiobacillus spp., Crenothrix spp., Clonothrix spp., Methylocystis spp., Methylocucumis spp., Methyloferula spp., Methylosinus spp., Methylocella spp., Methylocapsa spp., Methylacidiphilum spp., Methylacidimicrobium spp., Methylomirabilis spp., Methanoperedens spp., Mycolicibacterium spp., and combinations thereof; wherein the second microbe is a wild-type or a genetically modified mixotrophic bacteria selected from a group comprising Cupriavidus spp., Escherichia spp., Aquifex spp., Clostridium spp., Corynebacterium spp., Gordonia spp., Nocardia spp., Rhodobacter spp., Rhodopseudomonas spp., Rhodospirillum spp., Rhodococcus spp., Rhizobium spp., Thiocapsa spp., Pseudomonas spp., Hydrogenomonas spp., Hydrogenobacter spp., Hydrogenophilus spp., Hydrogenovibrio spp., Hydrogenothermus spp., Helicobacter spp., Xanthobacter spp., Hydrogenophaga spp., Bradyrhizobium spp., Ralstonia spp., Alcaligenes spp., Amycolata spp., Aquaspirillum spp., Arthrobacter spp., Azospirillum spp., Variovorax spp., Acidovorax spp., Bacillus spp., Calderobacterium spp., Derxia spp., Flavobacterium spp., Microcyclus spp., Mycobacterium spp., Paracoccus spp., Persephonella spp., Renobacter spp., Streptomyces spp., Thermocrinis spp., Wautersia spp., and combinations thereof.; wherein the method is carried out at a temperature of about 5°C to about 50°C at a pH of about 3 to 8; and wherein the culturing is carried out by batch fermentation, fed-batch fermentation, or continuous fermentation.

[0102] In some embodiments, the method comprises:- culturing Methylosinus trichosporium in presence of the methane (CH4), and subjecting the Methylosinus trichosporium to stress to produce an organic acid and hydrogen (H2); and- culturing the Methylosinus trichosporium and a Cupriavidus necator in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce thevalue-added product, wherein the Methylosinus trichosporium and Cupriavidus necator are present in a first / single bioreactor; wherein the methanotrophic bacteria utilizes the hydrogen in presence of CO2 to produce the value-added products and the second microbe utilizes the hydrogen or the organic acids or both, in presence of CO2 to produce the value-added products; wherein the method is carried out at a temperature of about 5°C to about 50°C at a pH of about 3 to 8; and wherein the culturing is carried out by batch fermentation.

[0102] The present disclosure also provides a method of culturing a microbe. While the subsequent embodiments focus on method of culturing a microbe, the features, and characteristics of the method of utilizing CH4 and CO2 including the parameters, the methanotrophic bacteria and the second microbe are as described by any of the embodiments above. For the sake of brevity, and avoiding repetition, each of those embodiments are not being reiterated here again. However, each of the said embodiments completely fall within the purview of the method of culturing described herein below.

[0103] In some embodiments, the method of culturing a microbe, comprises: a) growing a methanotrophic bacteria in presence of methane (CH4); b) subjecting said methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and c) providing CO2 to the methanotrophic bacteria, wherein the methanotrophic bacteria utilizes the hydrogen produced in step (b) and CO2 to produce value-added products; d) optionally, providing CO2 and organic acids and hydrogen (H2) produced in step b) to a second microbe to produce value-added products.

[0104] In some embodiments, the method of culturing a microbe, comprises: a) growing a methanotrophic bacteria in presence of methane (CH4);b) subjecting said methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and c) providing CO2 to the methanotrophic bacteria, wherein the methanotrophic bacteria utilizes the hydrogen produced in step (b) and CO2 to produce value-added products.

[0105] In some embodiments, the method of culturing a microbe, comprises: a) growing a wild type or recombinant Methylosinus trichosporium in presence of methane (CH4); b) subjecting said Methylosinus trichosporium to nutrient stress of limited Cu availability to produce organic acids and hydrogen (H2); and c) providing CO2 to the Methylosinus trichosporium, wherein Methylosinus trichosporium utilizes the hydrogen produced in step (b) and CO2 to produce value- added products.

[0106] In some embodiments, the method of culturing a microbe, comprises: a) growing a methanotrophic bacteria in presence of methane (CH4) in a first bioreactor; b) subjecting said methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and c) providing CO2 to the methanotrophic bacteria, wherein the methanotrophic bacteria utilizes the hydrogen produced in step (b) and CO2 to produce value-added products; and d) providing CO2 and organic acids and hydrogen (H2) produced in step b) to a second microbe to produce value-added products.

[0107] In some embodiments, the method of culturing a microbe, comprises: a) growing a methanotrophic bacteria in a first bioreactor in presence of methane (CH4) in a first bioreactor; b) subjecting said methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); andc) providing CO2 to the methanotrophic bacteria, wherein the methanotrophic bacteria utilizes the hydrogen produced in step (b) and CO2 to produce value-added products; and d) providing CO2 and organic acids and hydrogen (H2) produced in step b) to a second microbe in a second bioreactor to produce value-added products.

[0108] In some embodiments, the method of culturing a microbe, comprises: a) growing a wild type or recombinant Methylosinus trichosporium in a first bioreactor in presence of methane (CH4) in a first bioreactor; b) subjecting said Methylosinus trichosporium to nutrient stress of limited Cu availability to produce organic acids and hydrogen (H2); and c) providing CO2 to Methylosinus trichosporium, wherein the Methylosinus trichosporium utilizes the hydrogen produced in step (b) and CO2 to produce value- added products; and d) providing CO2 and organic acids and hydrogen (H2) produced in step b) Cupriavidus necatar in a second bioreactor to produce value-added products.

[0109] In some embodiments, the method of co-culturing comprises: a) growing a methanotrophic bacteria in presence of methane (CH4) in a bioreactor; b) subjecting said methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); c) providing CO2 to the methanotrophic bacteria, wherein the methanotrophic bacteria uses the hydrogen produced in step (b) and CO2 to produce value-added products; d) providing CO2, and organic acids and hydrogen (H2) produced in step b) to a mixotrophic bacteria in the bioreactor to produce value-added products.

[0110] In some embodiments, the method of co-culturing comprises: a) growing Methylosinus trichosporium in presence of methane (CH4) in a bioreactor; b) subjecting said Methylosinus trichosporium to stress to produce organic acids and hydrogen (H2);c) providing CO2 to Methylosinus trichosporium, wherein the Methylosinus trichosporium uses the hydrogen produced in step (b) and CO2 to produce value- added products; d) providing CO2, and organic acids and hydrogen (H2) produced in step b) Cupriavidus necatar in the bioreactor to produce value-added products.Recombinant methanotrophic bacteria

[0111] The present disclosure also provides recombinant microbes, particularly, recombinant or genetically modified methanotrophs. The recombinant methanotrophs of the present disclosure are able to utilize carbon dioxide apart from methane under specific cultivation conditions.

[0112] Methanotrophs have been reported to perform bioconversion of carbon dioxide to methanol as shown in the below Figure A.Figure A: Diagram illustrating co-utilization of methane and carbon dioxide by methanotrophsThis conversion is possible due to the reversible nature of steps from methanol (to formaldehyde to formate) to carbon dioxide (as can be seen the first step from methane to methanol is not reversible, whereas the downstream steps are reversible). Importantly, the bioconversion of carbon dioxide stops at the methanol step as methane to methanol step is not reversible. The wild-type organism has limited capacity to produce methanol, with the bottleneck being the supply of reducing equivalents. Moreover, methanol secretion has been shown to be proportional to the polyhydroxybutyrate content and external supply of a reducing source.

[0113] On the other hand, under nutrient stress or microaerobic cultivation, methanotrophs have also been shown to produce mixed organic acids through fermentation and release hydrogen. In the present disclosure, the inventors have developed a genetically engineered methanotroph thatallows co-utilization of carbon dioxide and methane under nutrient limited conditions. In the recombinant strain, genes related to redox equivalent recycling that facilitate sustained utilization of carbon dioxide and methane are targeted. Due to this redox recycling, hydrogen production or redox equivalents are supplied for downstream carboxylation reactions to drive carbon dioxide utilization. Genes related to increasing the supply of oxygen for achieving efficient methane utilization were also targeted.

[0114] Particularly, the following pathways or genes, either singly or in combination, are targeted to prepare recombinant methanotrophic bacteria of the present disclosure:For efficient utilization of methane under normal or O2 stress conditions, a homologous or heterologous bacteriohemerythrin gene is overexpressed to scavenge and / or increase O2 concentration inside the methanotroph. In addition, key genes involved in efficient methane utilization under O2 limiting conditions are considered for engineering;For NAD(P)H regeneration, homologous or heterologous genes / pathways for NAD(P)H synthesis or recycling are overexpressed;For increasing CO2 fixation, carboxylation systems (including carboxylases and carbonic anhydrases), central carbon metabolism, redox balance and regeneration systems are overexpressed;For reducing CO2 evolution, native formate dehydrogenase (FDH) genes are knocked out and a heterologous formate dehydrogenase that favors CO2 to formate production is introduced in methanotrophic bacteria.

[0115] In some embodiments, the recombinant methanotrophic bacteria comprises at least one gene modification selected from a group comprising: i. overexpressed homologous or heterologous bacteriohemerythrin gene; ii. overexpressed homologous or heterologous genes or pathways for NAD(P)H synthesis or recycling; iii. overexpressed genes encoding one or more enzymes selected from a group comprising carboxylation systems (including carboxylases and carbonic anhydrases), central carbon metabolism, redox balance and regeneration systems ; iv. knocked out native and a heterologous formate dehydrogenase (FDH) genes;or combinations thereof.

[0116] In some embodiments, the gene is selected from ccfdh (formate dehydrogenase H), fdhD (formate dehydrogenase accessory protein), fhs (formate-tetrahydrofolate ligase), zwf (glucose-6-phosphate dehydrogenase), ppc (phosphoenolpyruvate carboxylase), pgi (phosphoglucose isomerase), bhr (bacteriohemerythrin), hoxFUYHWI gene cluster, or combinations thereof.

[0117] In some embodiments, the recombinant methanotrophic bacterium comprises an overexpressed homologous or heterologous bacteriohemerythrin (bhr) gene that enhances oxygen tolerance and redox balance under methane-oxidizing and stress conditions.

[0118] In some embodiments, the recombinant methanotrophic bacterium comprises one or more overexpressed homologous or heterologous genes or pathways involved in NAD(P)H synthesis or recycling, including but not limited to zwf (glucose-6-phosphate dehydrogenase), and hoxFUYHWI (NAD(P)+-reducing hydrogenase gene cluster.

[0119] In some embodiments, the recombinant methanotrophic bacterium comprises overexpressed genes encoding enzymes of carboxylation systems, central carbon metabolism, or redox regeneration systems. Such genes include ppc (phosphoenolpyruvate carboxylase), pgi (phosphoglucose isomerase), and fhs (formate-tetrahydrofolate ligase).

[0120] In some embodiments, the gene is ccfdh (formate dehydrogenase H) having a nucleotide sequence as set forth in SEQ ID NO. 1 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 1.

[0121] In some embodiments, the gene is fdhD (formate dehydrogenase accessory protein) having a nucleotide sequence as set forth in SEQ ID NO. 2 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 2.

[0122] In some embodiments, the gene is fhs (formate-tetrahydrofolate ligase) having a nucleotide sequence as set forth in SEQ ID NO. 3 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 3.

[0123] In some embodiments, the gene is zwf (glucose-6-phosphate dehydrogenase) having a nucleotide sequence as set forth in SEQ ID NO. 4 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 4.

[0124] In some embodiments, the gene is ppc (phosphoenolpyruvate carboxylase) having a nucleotide sequence as set forth in SEQ ID NO. 5 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 5.

[0125] In some embodiments, the gene is pgi (phosphoglucose isomerase) having a nucleotide sequence as set forth in SEQ ID NO. 6 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 6.

[0126] In some embodiments, the gene is bhr (bacteriohemerythrin) having a nucleotide sequence as set forth in SEQ ID NO. 7 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 7.

[0127] In some embodiments, the gene is a hoxFUYHWI gene cluster, comprising a hoxF gene having a nucleotide sequence as set forth in SEQ ID NO. 8 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 8.

[0128] In some embodiments, the gene is a hoxFUYHWI gene cluster, comprising a hoxU gene having a nucleotide sequence as set forth in SEQ ID NO. 9 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 9.

[0129] In some embodiments, the gene is a hoxFUYHWI gene cluster, comprising a hoxY gene having a nucleotide sequence as set forth in SEQ ID NO. 10 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 10.

[0130] In some embodiments, the gene is a hoxFUYHWI gene cluster, comprising a hoxH gene having a nucleotide sequence as set forth in SEQ ID NO. 11 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 11.

[0131] In some embodiments, the gene is a hoxFUYHWI gene cluster, comprising a hoxW gene having a nucleotide sequence as set forth in SEQ ID NO. 12 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 12.

[0132] In some embodiments, the gene is a hoxFUYHWI gene cluster, comprising a hoxl gene having a nucleotide sequence as set forth in SEQ ID NO. 13 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 13.

[0133] In some embodiments, the recombinant methanotrophic bacterium comprises a gene knockout or deletion of native or heterologous formate dehydrogenase (FDH) genes to modulate carbon flux and redox balance.

[0134] In some embodiments, the recombinant methanotrophic bacterium comprises combinations of two or more of the above genetic modifications for enhanced methane oxidation, CO2 assimilation, and production of value-added compounds.

[0135] In some embodiments, the wild-type or recombinant methanotrophic bacteria is selected from a type I, type II, type X, or a Verrumicrobial methanotroph.

[0136] In some embodiments, the wild-type or recombinant methanotrophic bacteria is type I or type II or type X methanotroph belonging to genus selected from a group comprising Methylosinus spp. Methylococcus spp., Methylomonas spp., Methylobacter spp., Methylomicrobium spp., Halomonas spp., Methylosarcina spp., Methylocaldum spp., Methylogaea spp., Methylosoma spp., Methyloparacoccus spp., Methyloglobulus spp., Methyloprofundus spp., Methylomarinum spp., Methylovulum spp., Methylomagnum spp., Methylosphaera spp., Methylohalobius spp., Methylothermus spp., Methylomarinovum spp., Methylobacterium spp., Acidithiobacillus spp., Crenothrix spp., Clonothrix spp., Methylocystis spp., Methylocucumis spp., Methyloferula spp.,., Methylocella spp., Methylocapsa spp., Methylacidiphilum spp., Methylacidimicrobium spp., Methylomirabilis spp., Methanoperedens spp., Mycolicibacterium spp. an combinations thereof

[0137] In some embodiments, the wild-type or recombinant methanotrophic bacteria is type I or type II or type X methanotroph belonging to genus selected from a group comprising Methylococcus capsulatus, Methylococcus bovis, Methylococcus chroococcus, Methylococcus geothermalis, Methylococcus luteus, Methylococcus mesophilus, Methylococcus mobilis,Methylococcus thermophilus, Methylococcus vinelandii, Methylococcus whittenburyi, Methylomonas agile, Methylomonas albus, Methylomonas aurantiaca, Methylomonas aurea, Methylomonas defluvii, Methylomonas denitrificans, Methylomonas flagellata, Methylomonas fluvii, Methylomonas fodinarum, Methylomonas gracilis, Methylomonas koyamae, Methylomonas lenta, Methylomonas methanica, Methylomonas methanolica, Methylomonas methanooxidans, Methylomonas methylovora, Methylomonas montana, Methylomonas paludis, Methylomonas pelagica, Methylomonas rapida, Methylomonas rhizoryzae, Methylomonas rivi, Methylomonas rosea, Methylomonas rubra, Methylomonas scandinavica, Methylomonas subterranea, Methylobacter agilis, Methylobacter albus, Methylobacter alcaliphilus, Methylobacter capsulatus, Methylobacter chroococcum, Methylobacter luteus, Methylobacter marinus, Methylobacter modestohalophilus, Methylobacter pelagicus, Methylobacter psychrophilus, Methylobacter svalbardensis, Methylobacter tundripaludum, Methylobacter vinelandii, Methylobacter whittenburyi, Methylomicrobium agile, Methylomicrobium album, Methylomicrobium alcaliphilum, Methylomicrobium buryatense, Methylomicrobium japanense, Methylomicrobium kenyense, Methylomicrobium lacus, Methylomicrobium pelagicum, Halomonas pantelleriensis, Methylosarcina fibrata, Methylosarcina lacus, Methylosarcina quisquiliarum, Methylocaldum gracile, Methylocaldum marinum, Methylocaldum szegediense, Methylocaldum tepidum, Methylogaea oryzae, Methylosoma difficile, Methyloparacoccus murrellii, Methyloglobulus morosus, Methyloprofiundus sedimenti, Methylomarinum vadi, Methylovulum miyakonense, Methylovulum psychrotolerans, Methylomagnum ishizawai, Methylosphaera hansonii, Methylohalobius crimeensis, Methylothermus subterraneus, Methylothermus thermalis, Methylomarinovum caldicuralii, Methylomarinovum tepidoasis, Methylobacterium aminovorans, Methylobacterium thiocyanatum, Methylobacterium zatmanii, Acidithiobacillus ferrivorans, Methylobacterium aquaticum, Methylobacterium suomiense, Methylobacterium adhaesivum, Methylobacterium podarium, Methylobacterium variabile, Methylobacterium vinelandii, Methylobacterium hispanicum, Crenothrix polyspora, Clonothrix fusca, Methylocystis methanolicus, Methylocucumis oryzae, Methylogaea oryzae, Methylosarcina lacus, Methylosoma difficile, Methyloferula stellata, Methylosinus trichosporium, Methylocystis borborid, Methylocystis bryophila, Methylocystis echinoides, Methylocystis heyeri, Methylocystis hirsuta, Methylocystis iwaonis, Methylocystis minimus, Methylocystis parva, Methylocystis parvus, Methylocystis rosea, Methylocystis silviterrae, Methylocystis suflitae, Methylosinuspucelana, Methylosinus sporium, Methylocella palustris, Methylocella silvestris, Methylocella tundrae, Methylocapsa acidiphila, Methylocapsa aurea, Methylocapsa gorgona, Methylocapsa paisarum, Methyloferula stellata, Methylacidiphilum caldifontis, Methylacidiphilum fumariolicum, Methylacidiphilum infemorum, Methylacidiphilum kamchatkense, Methylacidimicrobium cyclopophantes, Methylacidimicrobium fagopyrum, Methylacidimicrobium tartarophylax, Methylacidimicrobium thermophilum, Methylomirabilis iodofontis, Methylomirabilis lanthanidiphila, Methylomirabilis limnetica, Methylomirabilis nitratireducens, Methylomirabilis oxyfera, Methylomirabilis oxygeniifera, Methylomirabilis sinica, Methylomirabilis tolerans, Methanoperedens ferrireducens, Methanoperedens manganicus, Methanoperedens manganireducens, Methanoperedens nitratireducens, Methanoperedens nitroreducens, Methanoperedens psychrophilus, and combinations thereof..

[0138] In some embodiments, a recombinant methanotrophic bacterial strain is engineered to co-express the ccfdh (formate dehydrogenase H) and fdhD (formate dehydrogenase accessory protein) genes. The ccfdh and fdhD sequences are operably linked under a constitutive promoter and introduced into M. trichosporium using a broad-host-range expression vector. The coexpression of these genes enables efficient reduction of carbon dioxide (CO2) to formate and enhances redox balance under nutrient-limited conditions.

[0139] In some embodiments, a recombinant Methylosinus trichosporium strain is engineered to co-express the ccfdh (formate dehydrogenase H) and fdhD (formate dehydrogenase accessory protein) genes. The ccfdh and fdhD sequences are operably linked under a constitutive promoter and introduced into M. trichosporium using a broad-host-range expression vector. The coexpression of these genes enables efficient reduction of carbon dioxide (CO2) to formate and enhances redox balance under nutrient-limited conditions.

[0140] In some embodiments, a recombinant methanotrophic bacterial strain is engineered to co-express the ccfdh (formate dehydrogenase H) gene having a nucleotide sequence as set forth in SEQ ID NO. 1 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 1; and fdhD (formate dehydrogenase accessory protein) gene having a nucleotide sequence as set forth in SEQ ID NO. 2 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 2. The ccfdh andfdhD sequences are operably linked under a constitutive promoter and introduced into M. trichosporium using a broad-host-range expression vector. The co-expression of these genes enables efficient reduction of carbon dioxide (CO2) to formate and enhances redox balance under nutrient-limited conditions.

[0141] In some embodiments, a recombinant Methylosinus trichosporium strain is engineered to co-express the ccfdh (formate dehydrogenase H) gene having a nucleotide sequence as set forth in SEQ ID NO. 1 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 1; and fdhD (formate dehydrogenase accessory protein) gene having a nucleotide sequence as set forth in SEQ ID NO. 2 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 2. The ccfdh and fdhD sequences are operably linked under a constitutive promoter and introduced into M. trichosporium using a broad-host-range expression vector. The co-expression of these genes enables efficient reduction of carbon dioxide (CO2) to formate and enhances redox balance under nutrient-limited conditions.

[0142] In some embodiments, a recombinant Methylosinus trichosporium strain is engineered to co-express the ccfdh (formate dehydrogenase H) gene having a nucleotide sequence as set forth in SEQ ID NO. 1; and fdhD (formate dehydrogenase accessory protein) gene having a nucleotide sequence as set forth in SEQ ID NO. 2. The ccfdh and fdhD sequences are operably linked under a constitutive promoter and introduced into M. trichosporium using a broad-host- range expression vector. The co-expression of these genes enables efficient reduction of carbon dioxide (CO2) to formate and enhances redox balance under nutrient-limited conditions.

[0143] In some embodiments, a recombinant methanotrophic bacterial strain comprises a dualgene operon containing fhs (formate-tetrahydrofolate ligase) and zwf (glucose-6-phosphate dehydrogenase). The fhs gene facilitates incorporation of formate- derived carbon via the folate cycle, while zwf provides enhanced NADPH regeneration through the pentose phosphate pathway. The combination of these two genes improves carbon assimilation and biosynthetic capacity in the recombinant strain.

[0144] In some embodiments, a recombinant M. trichosporium strain comprises a dual-gene operon containing fhs (formate-tetrahydrofolate ligase) and zwf (glucose-6-phosphate dehydrogenase). The fits gene facilitates incorporation of formate- derived carbon via the folate cycle, while zwf provides enhanced NADPH regeneration through the pentose phosphate pathway. The combination of these two genes improves carbon assimilation and biosynthetic capacity in the recombinant strain.

[0145] In some embodiments, a recombinant methanotrophic bacterial strain comprises a dualgene operon containing / (formate-tetrahydrofolate ligase) gene having a nucleotide sequence as set forth in SEQ ID NO. 3 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 3; and zwf (glucose-6-phosphate dehydrogenase) gene having a nucleotide sequence as set forth in SEQ ID NO. 4 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 4. The fhs gene facilitates incorporation of formate-derived carbon via the folate cycle, while zwf provides enhanced NADPH regeneration through the pentose phosphate pathway. The combination of these two genes improves carbon assimilation and biosynthetic capacity in the recombinant strain.

[0146] In some embodiments, a recombinant M. trichosporium strain comprises a dual-gene operon containing fhs (formate-tetrahydrofolate ligase) gene having a nucleotide sequence as set forth in SEQ ID NO. 3 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 3; and zwf (glucose-6-phosphate dehydrogenase) gene having a nucleotide sequence as set forth in SEQ ID NO. 4 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 4. The fhs gene facilitates incorporation of formate-derived carbon via the folate cycle, while zwf provides enhanced NADPH regeneration through the pentose phosphate pathway. The combination of these two genes improves carbon assimilation and biosynthetic capacity in the recombinant strain.

[0147] In some embodiments, a recombinant M. trichosporium strain comprises a dual-gene operon containing fhs (formate-tetrahydrofolate ligase) gene having a nucleotide sequence as set forth in SEQ ID NO. 3; and zwf (glucose-6-phosphate dehydrogenase) gene having a nucleotide sequence as set forth in SEQ ID NO. 4. The fhs gene facilitates incorporation of formate-derived carbon via the folate cycle, while zw / provides enhanced NADPH regeneration through the pentosephosphate pathway. The combination of these two genes improves carbon assimilation and biosynthetic capacity in the recombinant strain.

[0148] In some embodiments, a recombinant methanotrophic bacterial strain comprises ppc (phosphoenolpyruvate carboxylase) gene under a constitutive promoter. Expression of ppc enables direct fixation of carbon dioxide into oxaloacetate, thereby increasing the pool of tricarboxylic acid (TCA) cycle intermediates such as malate and aspartate, and improving overall carbon flux toward biomass production.

[0149] In some embodiments, a recombinant M. trichosporium strain comprises ppc (phosphoenolpyruvate carboxylase) gene under a constitutive promoter. Expression of ppc enables direct fixation of carbon dioxide into oxaloacetate, thereby increasing the pool of tricarboxylic acid (TCA) cycle intermediates such as malate and aspartate, and improving overall carbon flux toward biomass production.

[0150] In some embodiments, a recombinant methanotrophic bacterial strain comprises ppc (phosphoenolpyruvate carboxylase) gene having a nucleotide sequence as set forth in SEQ ID NO. 5 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 5, under a constitutive promoter. Expression of ppc enables direct fixation of carbon dioxide into oxaloacetate, thereby increasing the pool of tricarboxylic acid (TCA) cycle intermediates such as malate and aspartate, and improving overall carbon flux toward biomass production.

[0151] In some embodiments, a recombinant M. trichosporium strain comprises ppc (phosphoenolpyruvate carboxylase) gene having a nucleotide sequence as set forth in SEQ ID NO.5 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 5, under a constitutive promoter. Expression of ppc enables direct fixation of carbon dioxide into oxaloacetate, thereby increasing the pool of tricarboxylic acid (TCA) cycle intermediates such as malate and aspartate, and improving overall carbon flux toward biomass production.

[0152] In some embodiments, a recombinant M. trichosporium strain comprises ppc (phosphoenolpyruvate carboxylase) gene having a nucleotide sequence as set forth in SEQ ID NO.5, under a constitutive promoter. Expression of ppc enables direct fixation of carbon dioxide into oxaloacetate, thereby increasing the pool of tricarboxylic acid (TCA) cycle intermediates such as malate and aspartate, and improving overall carbon flux toward biomass production.

[0153] In some embodiments, a recombinant methanotrophic bacterial strain is engineered to co-express ccfdh (formate dehydrogenase H) and pgi (phosphoglucose isomerase) genes. The ccfdh gene enables formate production from CO2, while pgi supports downstream carbon routing through glycolysis. The combined expression enhances carbon flow through central metabolism and improves growth and substrate utilization efficiency under mixed CH4 and CO2 feed conditions.

[0154] In some embodiments, a recombinant M. trichosporium strain is engineered to coexpress ccfdh (formate dehydrogenase H) and pgi (phosphoglucose isomerase) genes. The ccfdh gene enables formate production from CO2, while pgi supports downstream carbon routing through glycolysis. The combined expression enhances carbon flow through central metabolism and improves growth and substrate utilization efficiency under mixed CH4 and CO2 feed conditions.

[0155] In some embodiments, a recombinant methanotrophic bacterial strain is engineered to co-express ccfdh (formate dehydrogenase H) gene having a nucleotide sequence as set forth in SEQ ID NO. 1 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 1; and pgi (phosphoglucose isomerase) gene having a nucleotide sequence as set forth in SEQ ID NO. 6 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 6. The ccfdh gene enables formate production from CO2, while pgi supports downstream carbon routing through glycolysis. The combined expression enhances carbon flow through central metabolism and improves growth and substrate utilization efficiency under mixed CH4 and CO2 feed conditions.

[0156] In some embodiments, a recombinant M. trichosporium strain is engineered to coexpress ccfdh (formate dehydrogenase H) gene having a nucleotide sequence as set forth in SEQ ID NO. 1 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 1; and pgi (phosphoglucose isomerase) gene having a nucleotide sequence as set forth in SEQ ID NO. 6 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 6. The ccfdh gene enables formateproduction from CO2, while pgi supports downstream carbon routing through glycolysis. The combined expression enhances carbon flow through central metabolism and improves growth and substrate utilization efficiency under mixed CH4 and CO2 feed conditions.

[0157] In some embodiments, a recombinant M. trichosporium strain is engineered to coexpress ccfdh (formate dehydrogenase H) gene having a nucleotide sequence as set forth in SEQ ID NO. 1; and pgi (phosphoglucose isomerase) gene having a nucleotide sequence as set forth in SEQ ID NO. 6. The ccfdh gene enables formate production from CO2, while pgi supports downstream carbon routing through glycolysis. The combined expression enhances carbon flow through central metabolism and improves growth and substrate utilization efficiency under mixed CH4 and CO2 feed conditions.

[0158] In some embodiments, a recombinant methanotrophic bacterial strain overexpresses the bhr (bacteriohemerythrin) gene under a constitutive promoter. The Bhr protein, an oxygen-binding redox protein, enhances oxygen tolerance and maintains redox homeostasis during methane oxidation and transient oxygen limitation. Overexpression of bhr results in improved cell viability, sustained methane monooxygenase activity, and higher methanol conversion efficiency.

[0159] In some embodiments, a recombinant M. trichosporium strain overexpresses the bhr (bacteriohemerythrin) gene under a constitutive promoter. The Bhr protein, an oxygen-binding redox protein, enhances oxygen tolerance and maintains redox homeostasis during methane oxidation and transient oxygen limitation. Overexpression of bhr results in improved cell viability, sustained methane monooxygenase activity, and higher methanol conversion efficiency.

[0160] In some embodiments, a recombinant methanotrophic bacterial strain overexpresses the bhr (bacteriohemerythrin) gene having a nucleotide sequence as set forth in SEQ ID NO. 7 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 7, under a constitutive promoter. The Bhr protein, an oxygen-binding redox protein, enhances oxygen tolerance and maintains redox homeostasis during methane oxidation and transient oxygen limitation. Overexpression of bhr results in improved cell viability, sustained methane monooxygenase activity, and higher methanol conversion efficiency.

[0161] In some embodiments, a recombinant M. trichosporium strain overexpresses the bhr (bacteriohemerythrin) gene having a nucleotide sequence as set forth in SEQ ID NO. 7 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 7, under a constitutive promoter. The Bhr protein, an oxygen-binding redox protein, enhances oxygen tolerance and maintains redox homeostasis during methane oxidation and transient oxygen limitation. Overexpression of bhr results in improved cell viability, sustained methane monooxygenase activity, and higher methanol conversion efficiency.

[0162] In some embodiments, a recombinant M. trichosporium strain overexpresses the bhr (bacteriohemerythrin) gene having a nucleotide sequence as set forth in SEQ ID NO. 7, under a constitutive promoter. The Bhr protein, an oxygen-binding redox protein, enhances oxygen tolerance and maintains redox homeostasis during methane oxidation and transient oxygen limitation. Overexpression of bhr results in improved cell viability, sustained methane monooxygenase activity, and higher methanol conversion efficiency.

[0163] In some embodiments, a recombinant methanotrophic bacterial strain expresses a heterologous hoxFUYHWI gene cluster encoding an oxygen-tolerant, NAD(P)+-reducing hydrogenase derived from Cupriavidus necator. The gene cluster is operably linked under a suitable promoter and expressed to couple hydrogen oxidation with NAD(P)H regeneration under microaerobic conditions. This modification enhances redox balance, cofactor regeneration, and overall carbon assimilation efficiency.

[0164] In some embodiments, a recombinant M. trichosporium strain expresses a heterologous hoxFUYHWI gene cluster encoding an oxygen-tolerant, NAD(P)+-reducing hydrogenase derived from Cupriavidus necator. The gene cluster is operably linked under a suitable promoter and expressed to couple hydrogen oxidation with NAD(P)H regeneration under microaerobic conditions. This modification enhances redox balance, cofactor regeneration, and overall carbon assimilation efficiency.

[0165] In some embodiments, a recombinant methanotrophic bacterial strain expresses a heterologous hoxFUYHWI gene cluster encoding an oxygen-tolerant, NAD(P)+-reducing hydrogenase derived from Cupriavidus necator. The gene cluster comprises i) hoxF gene havinga nucleotide sequence as set forth in SEQ ID NO. 8 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 8, ii) hoxU gene having a nucleotide sequence as set forth in SEQ ID NO. 9 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 9, iii) hoxY gene having a nucleotide sequence as set forth in SEQ ID NO. 10 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 10, iv) hoxH gene having a nucleotide sequence as set forth in SEQ ID NO. 11 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 11, v) hoxW gene having a nucleotide sequence as set forth in SEQ ID NO. 12 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 12, and vi) hoxl gene having a nucleotide sequence as set forth in SEQ ID NO. 13 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 13. The gene cluster is operably linked under a suitable promoter and expressed to couple hydrogen oxidation with NAD(P)H regeneration under microaerobic conditions. This modification enhances redox balance, cofactor regeneration, and overall carbon assimilation efficiency.

[0166] In some embodiments, a recombinant M. trichosporium strain expresses a heterologous hoxFUYHWI gene cluster encoding an oxygen-tolerant, NAD(P)+-reducing hydrogenase derived from Cupriavidus necator. The gene cluster comprises i) hoxF gene having a nucleotide sequence as set forth in SEQ ID NO. 8 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 8, ii) hoxU gene having a nucleotide sequence as set forth in SEQ ID NO. 9 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 9, iii) hoxY gene having a nucleotide sequence as set forth in SEQ ID NO. 10 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 10, iv) hoxH gene having a nucleotide sequence as set forth in SEQ ID NO. 11 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 11, v) hoxW gene having a nucleotide sequence as set forth in SEQ ID NO. 12 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 12, and vi) hoxl gene having a nucleotide sequence as set forth in SEQ ID NO. 13 or a nucleotide sequence having at least 80% identity to SEQ ID NO. 13. The gene cluster is operably linked under a suitable promoter and expressed to couple hydrogen oxidation with NAD(P)H regeneration under microaerobic conditions. This modification enhances redox balance, cofactor regeneration, and overall carbon assimilation efficiency.

[0167] In some embodiments, a recombinant M. trichosporium strain expresses a heterologous hoxFUYHWI gene cluster encoding an oxygen-tolerant, NAD(P)+-reducing hydrogenase derived from Cupriavidus necator. The gene cluster comprises i) hoxF gene havinga nucleotide sequence as set forth in SEQ ID NO. 8 ii) hoxU gene having a nucleotide sequence as set forth in SEQ ID NO. 9 iii) hoxY gene having a nucleotide sequence as set forth in SEQ ID NO. 10 iv) hoxH gene having a nucleotide sequence as set forth in SEQ ID NO. 11, v) hoxW gene having a nucleotide sequence as set forth in SEQ ID NO. 12, and vi) hoxl gene having a nucleotide sequence as set forth in SEQ ID NO. 13. The gene cluster is operably linked under a suitable promoter and expressed to couple hydrogen oxidation with NAD(P)H regeneration under microaerobic conditions. This modification enhances redox balance, cofactor regeneration, and overall carbon assimilation efficiency.

[0168] In some embodiments, the promoter used for synthesis of recombinant methanotrophic bacteria is selected from pmxaF, 3-hexulose-6-phosphate synthase promoter (hps), Formal dehyde- activating enzyme promoter (fae2), c54(RpoN)-dependent promoter, or c70(RpoD)-dependent promoter.

[0169] In some embodiments, the promoter used for synthesis of recombinant methanotrophic bacteria is pmxaF having a nucleotide sequence as set forth in SEQ ID NO. 14.

[0170] In some embodiments, the promoter used for synthesis of recombinant methanotrophic bacteria is hps having a nucleotide sequence as set forth in SEQ ID NO. 15.

[0171] In some embodiments, the promoter used for synthesis of recombinant methanotrophic bacteria is fae2 having a nucleotide sequence as set forth in SEQ ID NO. 16.

[0172] In some embodiments, the promoter used for synthesis of recombinant methanotrophic bacteria is c54having a nucleotide sequence as set forth in SEQ ID NO. 17.

[0173] In some embodiments, the promoter used for synthesis of recombinant methanotrophic bacteria is G70having a nucleotide sequence as set forth in SEQ ID NO. 18.

[0174] In some embodiments, the gene modification is introduced in a methanotrophic bacteria with a vector comprising one or more expression cassettes encoding the gene as above.

[0175] In some embodiments, the vector comprises one or more expression cassettes encoding genes selected from ccfdh,fdhD,jhs, zwf ppc, pgi, bhr, hoxFUYHWI or combinations thereof.

[0176] In some embodiments, the recombinant methanotrophic bacteria modified by integrating heterologous pathway(s) / gene modifications as described above exhibit the following traits / properties:- better utilization of CH4 under normal conditions and / or stress,- efficient regeneration / t / e novo synthesis of NAD(P)H and other redox equivalents,- better (re-)utilization of CO2, and- production of novel metabolites / target molecules leveraging pathways upregulated during CO2-CH4 co-utilization and channeling of the relevant metabolites from these pathways.VectorThe present disclosure also provides a single or multiple vectors. In some embodiments, the present disclosure provides a single vector or a single co-expression vector. In some embodiments, the present disclosure provides multiple vectors.

[0177] While the subsequent embodiments focus on vectors, the features, and characteristics of the recombinant methanotrophic bacteria and the second microbe are as described by any of the embodiments above. For the sake of brevity, and avoiding repetition, each of those embodiments are not being reiterated here again. However, each of the said embodiments completely fall within the purview of the vectors described herein below.

[0178] More particularly, in some embodiments, the vector of the present disclosure comprises one or more expression cassette having one or more gene for overexpression in a microbe, said gene selected from a group comprising:- homologous or heterologous bacteriohemerythrin gene;- homologous or heterologous genes or pathways for NAD(P)H synthesis or recycling;- genes encoding one or more enzymes selected from a group comprising carboxylation systems (including carboxylases and carbonic anhydrases), central carbon metabolism, redox balance, and regeneration systems.

[0179] In some embodiments, the vector comprises one or more expression cassette comprising gene selected from ccfdh (formate dehydrogenase H), fdhD (formate dehydrogenase accessory protein), fits (formate-tetrahydrofolate ligase), zwf (glucose-6-phosphate dehydrogenase), ppc (phosphoenolpyruvate carboxylase), pgi (phosphoglucose isomerase), bhr (bacteriohemerythrin), hoxFUYHWI gene cluster, or combinations thereof.

[0180] In some embodiments, the vector comprises one or more expression cassettes comprising a gene selected from: ccfdh gene having a nucleotide sequence as set forth in SEQ ID NO: 1, or a nucleotide sequence having at least 80% identity to SEQ ID NO. 1 ;- fdhD gene having a nucleotide sequence as set forth in SEQ ID NO: 2, or a nucleotide sequence having at least 80% identity to SEQ ID NO. 2;- fhs gene having a nucleotide sequence as set forth in SEQ ID NO: 3, or a nucleotide sequence having at least 80% identity to SEQ ID NO. 3;- zwf gene having a nucleotide sequence as set forth in SEQ ID NO: 4, or a nucleotide sequence having at least 80% identity to SEQ ID NO. 4;- ppc gene having a nucleotide sequence as set forth in SEQ ID NO: 5, or a nucleotide sequence having at least 80% identity to SEQ ID NO. 5;- pgi gene having a nucleotide sequence as set forth in SEQ ID NO: 6, or a nucleotide sequence having at least 80% identity to SEQ ID NO. 6;bhr gene having a nucleotide sequence as set forth in SEQ ID NO: 7, or a nucleotide sequence having at least 80% identity to SEQ ID NO. 7; and hoxFUYHWI gene cluster, wherein the hoxF, hoxU, hoxY, hoxH, hoxW, and hoxl genes have nucleotide sequences as set forth in SEQ ID NOs: 8 to 13, respectively, or nucleotide sequences having at least 80% identity to SEQ ID Nos: 8 to 13; or any combination thereof.

[0181] The present disclosure also provides a method of developing a recombinant methanotrophic bacteria, comprising:- transforming a wild-type methanotrophic bacteria with vector as described in embodiments above; and / or- subjecting said wild-type methanotrophic bacteria to random mutagenesis, or site directed mutagenesis, or both.System

[0182] The present disclosure also refers to a system for utilizing methane and carbon dioxide and a system for culturing a microbe.

[0183] While the subsequent embodiments focus on system, the features, and characteristics of the method, methanotrophic bacteria and second microbe - are as described by any of the embodiments above. For the sake of brevity, and avoiding repetition, each of those embodiments are not being reiterated here again. However, each of the said embodiments completely fall within the purview of the system described below.

[0184] More particularly, the present disclosure provides a system for utilizing methane (CH4) and carbon dioxide (CO2), comprising:- a first bioreactor having a methanotrophic bacteria;- a second bioreactor comprising a second microbe; wherein said first bioreactor comprises a first tube coupled to the body of the first bioreactor configured to supply CH4;wherein said first bioreactor comprises a second tube coupled to the body of the first bioreactor configured to supply CO2; wherein the first bioreactor and the second bioreactor are attached to each other by a third tube configured to supply organic acid and H2 from the first bioreactor to the second bioreactor; and wherein said system utilizes the CH4 and CO2 by performing the method as claimed in claims 1 to 12, wherein the method comprises the steps of:- culturing a methanotrophic bacteria in the first bioreactor in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and- culturing the methanotrophic bacteria in the first bioreactor and a second microbe in a second bioreactor in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce the value-added product; wherein the organic acid and hydrogen from the first bioreactor is introduced into the second bioreactor and utilized by the second microbe to produce the value- added product.

[0185] In some embodiments of the system, the first and second bioreactor are selected from a group comprising a stirred tank reactor, an airlift reactor, a bubble column reactor, or a plug flow reactor.The present disclosure also refers to use of recombinant methanotrophic bacteria for co-utilization of methane (CH4) and carbon dioxide (CO2).In some embodiments, present disclosure provides use of the recombinant methanotrophic bacterium as defined above for the co-utilization of methane (CH4) and carbon dioxide (CO2) to produce one or more value-added products.In some embodiments, present disclosure provides use of the recombinant methanotrophic bacterium as defined above for converting methane (CH4) and carbon dioxide (CO2) into organic acids, alcohols, polyhydroxyalkanoates, lipids, amino acids, or combinations thereof.In some embodiments, present disclosure provides use of the recombinant methanotrophic bacterium as defined above in a method utilizing methane (CH4) and carbon dioxide (CO2) to produce value-added product, comprising:- culturing the recombinant methanotrophic bacterium in presence of the methane (CH4), and subjecting the recombinant methanotrophic bacterium to stress to produce an organic acid and hydrogen (H2); and- culturing the recombinant methanotrophic bacteria in presence of carbon dioxide (CO2), and the hydrogen to produce the value-added product.In some embodiments, present disclosure provides use of the recombinant methanotrophic bacterium as defined above in a method utilizing methane (CH4) and carbon dioxide (CO2) to produce value-added product, comprising:- culturing a recombinant methanotrophic bacteria in a first bioreactor in presence of methane (CH4), and subjecting the recombinant methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and- culturing the recombinant methanotrophic bacteria in the first bioreactor and a second microbe in a second bioreactor in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce the value-added product; wherein the organic acid and hydrogen from the first bioreactor is introduced into the second bioreactor and utilized by the second microbe.In some embodiments, present disclosure provides use of the recombinant methanotrophic bacterium as defined above in a method utilizing methane (CH4) and carbon dioxide (CO2) to produce value-added product, comprising:- culturing a recombinant methanotrophic bacteria in presence of the methane (CH4), and subjecting the recombinant methanotrophic bacteria to stress to produce an organic acid and hydrogen (H2); and- culturing the recombinant methanotrophic bacteria and a second microbe in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce the value-added product, wherein the recombinant methanotrophic bacteria and the second microbe are present in a first / single bioreactor.It is to be understood that the foregoing description is illustrative and not limiting. While considerable emphasis has been placed herein on particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein.Descriptions of well-known / conventional methods / steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above-described embodiments, and in order to illustrate the embodiments of the present disclosure, certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments may be practiced and to further enable those of skill in the art to practice the embodiments. Accordingly, following examples should not be construed as limiting the scope of the embodiments herein.EXAMPLES

[0186] While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scopeof the disclosure. The disclosure is not limited to the described embodiments, versions, or examples, which are included to enable a person having ordinary skill in the art to make and use the disclosure when combined with information and knowledge available to the person having ordinary skill in the art.Source and origin of Biological material used in the experiments:Methylosinus trichosporium was obtained from Prof. Cohn Murrell's lab at the University of Norwich, UK.Cupriavidus necator was obtained from DSMZ culture collection center, Germany.Example 1: Method of utilizing methane and carbon dioxide (CO2)Methylosinus trichosporium was initially cultivated with methane (CH4) as the sole carbon and energy source using conventional methanotrophic culture techniques. The strain was subsequently subjected to nutrient stress by limiting copper availability to evaluate the effect of carbon dioxide (CO2) supplementation on growth and metabolite secretion.Experiment: About 5L of stirred tank reactor was filled with about 4L of NMS media composition and was inoculated with a starter culture of trichosporium. The pH was controlled at about 7.0 throughout the cultivation by automatic addition of acid or base as required. A gaseous mixture comprising methane and carbon dioxide in a range of about 100:0 or 80:20 to 40:60 was continuously supplied to the reactor. Agitation speed was varied between about 200 rpm and 1000 rpm during the cultivation period. The culture was maintained for about 135 hours under these conditions.Cultures were grown in nitrate mineral salts (NMS) medium formulated with or without copper (Cu) supplementation. Methane (CH4) was provided as the primary carbon source, and selected cultures were additionally supplied with carbon dioxide (CO2) to assess its effect under nutrientlimited (Cu-deficient) conditions.The final 1 x NMS medium (I L) was prepared by combining 100 mb of 10x NMS salts solution, 1 mL each of 1000 / stock solutions of Fe-EDTA, sodium molybdate, trace elements, and copper sulfate, and making up the final volume to 1 L with Milli-Q water. The 10x NMS salts stocksolution contained 10 g / L potassium nitrate (KNO3), 10 g / L magnesium sulfate heptahydrate (MgSO4 7ILO), and 2 g / L calcium chloride dihydrate (CaCE 2EEO). The Fe-EDTA 1000 stock solution contained 3.8 g / L Fe-EDTA; the sodium molybdate 1000 stock contained 260 mg / L Na2MoO4; and the copper sulfate 1000 stock contained 250 mg / L CuSCL 5EEO. The trace elements 1000 stock solution comprised 500 mg / L FeSCE, 400 mg / L ZnSCL 7EEO, 15 mg / L H3BO3, 50 mg / L C0CI2 6H2O, 250 mg / L Na-EDTA, 20 mg / L MnCE 4ILO, and 10 mg / L NiCE 6H2O. The NMS medium thus prepared was sterilized by autoclaving according to standard procedures and used for the cultivation of M. trichosporium.For bioreactor growth experiments, the inoculum was cultivated either in NMS medium containing copper (Cu) or in copper-deficient medium (NMS-Cu, prepared by complete omission of CuSCh). For certain nutrient stress experiments, the inoculum as well as the initial culture were first grown in NMS medium; following sufficient biomass accumulation, nutrient stress was induced by either gradual replacement of the culture medium with the desired stress medium or by pelleting and resuspension of the cells in the desired fresh medium.Cultures were maintained under controlled temperature and aeration conditions suitable for M. trichosporium growth. Growth was monitored by measuring optical density at 600 nm (ODeoo) at regular intervals for about 140 hours.As shown in Figure 4, CEE-grown M. trichosporium exhibited a marked increase in growth upon CO2 supplementation under copper-limited (NMS-Cu) conditions compared to cultures without CO2 and under copper-limited (NMS-Cu) conditions. In contrast, such growth enhancement was not observed in copper-sufficient NMS medium (data not shown). These results indicate that nutrient stress (copper limitation) induces CO2 utilization and sequestration, which contributes to enhanced growth phenotype under nutrient-limiting conditions.The culture supernatants were then collected at defined time intervals (24, 48, 72, 96, and 120 hours) and analyzed for secretion of organic acid content using standard quantification assays.As illustrated in Figure 5, CCh-supplemented, CEE-grown M. trichosporium cells under copper- limited conditions exhibited significantly higher secretion of organic acids as compared to M. trichosporium cells grown with CEL, CO2 supplementation and copper-sufficient conditions. For instance, in the CCh-supplemented, CEE-grown M. trichosporium cells under copper-limitedconditions the succinic acid accumulation reached approximately 40 mg / L within 24 hours, and acetic acid levels increased progressively, reaching ~2.5 mg / L within 120 hours.These results demonstrate that AT. trichosporium exhibits enhanced CO2 sequestration and organic acid secretion under nutrient stress conditions (Cu limitation). The findings suggest that nutrient limitation triggers redirection of carbon flux toward organic acid biosynthesis, thereby facilitating metabolic adaptation and CO2 fixation in methanotrophs.Example 2: Synthesis of vectors and recombinant methanotrophsThe given experimental protocol was followed to prepare the following recombinant methanotrophs: a) Construction of Broad-Host-Range (BHR) Vectors via Gibson AssemblyBroad-host-range (BHR) plasmid constructs containing one or more genes of interest were prepared using a Gibson Assembly-based cloning strategy.A BHR plasmid backbone containing replication and maintenance elements suitable for both Escherichia coli cloning strains and methanotroph hosts was linearized either by polymerase chain reaction (PCR) amplification or by digestion with appropriate restriction enzyme(s). The linearized vector DNA was purified using a commercially available DNA purification kit, according to the manufacturer ’ s instructions .Target gene(s) were amplified by PCR using primers designed with 15-30 base pair overlap regions homologous to the termini of the linearized vector. Amplified products were verified by agarose gel electrophoresis and purified prior to assembly.Equimolar concentrations of the linearized plasmid backbone and purified PCR products were combined in a Gibson Assembly reaction mixture and incubated at 50°C for 1 hour to facilitate seamless joining of overlapping DNA fragments. The assembled products were transformed into chemically competent A. coli cells (DH5a or XL-1 Blue) and plated on Luria-Bertani (LB) agar containing kanamycin for selection. Plates were incubated overnight at 37°C.Resulting colonies were screened by colony PCR using vector- or insert-specific primers. Positive clones were confirmed by restriction digestion and / or Sanger sequencing to verify correctassembly and orientation of the insert. Verified recombinant plasmids were propagated in / ■ / coli, purified using a plasmid isolation kit, and stored at -20°C for subsequent transformation experiments.For conjugation-based transformation experiments, the verified BHR plasmids were introduced into E. coli SI 7-1 cells, which served as donor strains for plasmid transfer into methanotrophs. b) Conjugation-Based TransformationA recipient culture of Methylosinus trichosporium was prepared by spreading a loopful of an existing culture onto nitrate mineral salts (NMS) protease peptone agar plates and incubating at 30°C under an anaerobic atmosphere supplemented with 20% (v / v) methane for approximately 24 hours.Donor E. coli S 17- 1 cells harboring the BHR plasmid construct of interest were plated on LB agar containing the corresponding antibiotic and incubated overnight at 37°C. A loopful of the donor cells was then transferred to the recipient M. trichosporium culture on a mating plate. The mixed culture was spread evenly and incubated at 30°C under an anaerobic atmosphere supplemented with 20% (v / v) methane for approximately 48 hours to facilitate conjugation.Following incubation, cells from the mating plate were transferred to NMS agar plates containing kanamycin and nalidixic acid to select for transformed M. trichosporium colonies. Plates were incubated at 30°C in an atmosphere containing 20% (v / v) methane.Transformants typically appeared after 2-3 weeks of incubation. The presence of the desired recombinant construct in AT. trichosporium was confirmed by gene-specific PCR and by assessing antibiotic resistance corresponding to the selective marker encoded on the BHR vector. Standard Kanamycin selection, traJ, trfA, OriV and Ori sequences were present in the BHR vector used for M. trichosporium transformations.In the present experiment, 5 different promoters were used in the BHR vectors - pmxaF, 3- hexulose-6-phosphate synthase promoter (hps), formaldehyde-activating enzyme promoter (fae2), G54(RpoN)-dependent promoter, and c70(RpoD)-dependent promoter.The above protocol was followed for the synthesis of the following recombinants:1. Recombinant methanotroph expressing ccfdh + fdhD genes - A recombinant Methylosinus trichosporium strain was engineered to co-express the ccfdh (formate dehydrogenase H) and fdhD (formate dehydrogenase accessory protein) genes. The ccfdh and fdhD sequences were operably linked under a constitutive promoter and introduced into M. trichosporium using a broad-host-range expression vector.2. Recombinant methanotroph expressing fhs + zwf genes : A vector with dual-gene operon containing fhs (formate-tetrahydrofolate ligase) and zwf (glucose-6-phosphate dehydrogenase) was constructed and introduced into M. trichosporium. The fhs gene enabled incorporation of formate-derived carbon via the folate cycle, while zwf provided enhanced NADPH generation through the pentose phosphate pathway.3. Recombinant methanotroph expressing ppc gene: The ppc gene encoding phosphoenolpyruvate carboxylase was heterologously expressed in M. trichosporium under a constitutive promoter.4. Recombinant methanotroph expressing ccfdh + pgi genes: A construct containing ccjdhi ormate dehydrogenase H) and pgi (phosphoglucose isomerase) was cloned into a vector and transformed into M. trichosporium.5. Recombinant methanotroph expressing bhr gene: A homologous construct carrying the bhr (bacteriohemerythrin) gene from AT. trichosporium was overexpressed under a constitutive promoter. The Bhr protein, known for its oxygen-binding property, facilitates maintenance of redox balance during transient oxygen exposure.6. Recombinant methanotroph expressing hoxFUYHWI gene cluster: A heterologous gene cluster (hoxFUYHWI) encoding an Ch-tolerant, NAD(P)+-reducing hydrogenase derived from Cupriavidus necator was cloned under a promoter and expressed in M. trichosporium to couple hydrogen oxidation with NAD(P)H regeneration under microaerobic conditions.All recombinant methanotrophic bacterial strains were prepared and tested for production of the value-added product in accordance with the methods described herein. The recombinant methanotrophic bacterial strains outperformed wild type strain for CCh sequestration.Example 3: Method of utilizing methane (CHA and carbon dioxideIn the first bioreactor, a recombinant M. trichosporium strain of Example 2 was cultured under nutrient-limited (Cu-limited) conditions with methane (CH4) as the primary carbon source andsupplemental carbon dioxide (CO2). The culture was maintained in a 5 L stirred-tank bioreactor under controlled temperature, pH, and aeration parameters appropriate for growth of the recombinant methanotroph. During cultivation, the recombinant M. trichosporium secreted measurable amounts of organic acids, such as succinic acid and acetic acid, into the growth medium.At the end of the cultivation phase, the spent culture medium containing secreted organic acids and other soluble metabolites was harvested by centrifugation to remove residual biomass and obtain a clarified supernatant or a spent medium.The spent medium obtained from the first bioreactor was introduced into a second bioreactor containing a culture of C. necator. The second bioreactor was further supplemented with CO2 and H2 to support autotrophic metabolism and growth.Cupriavidus necator exhibited enhanced growth kinetics upon supplementation with the spent methanotroph medium. The observed growth enhancement correlated with the presence of organic acids secreted from the M. trichosporium culture, indicating effective carbon recycling between the two bioreactor systems.Example 4: Method of utilizing methane and carbon dioxide (CO2) by co-culturingResults of example 1 demonstrated that Methylosinus trichosporium is capable of sequestering carbon dioxide (CO2) and channeling the fixed carbon toward the synthesis and secretion of organic acids, including succinate, acetate, and formate. Cupriavidus necator is a facultative autotroph capable of utilizing CO2 as the sole carbon source under autotrophic conditions, and it also exhibits heterotrophic growth on reduced organic acids such as acetate, succinate, and formate.In the present experiment, M. trichosporium and C. necator were co-cultured under methane (CH4) and CCh-enriched conditions at varying ratios.Co-culture experiments were conducted in 250 mb flasks containing nitrate mineral salts (NMS) medium supplemented with bicarbonate buffer and maintained with CH4 feeding. CO2 was provided in the form of sodium bicarbonate (5.5 g / L final concentration). The initial inoculation optical densities (ODeoo) of trichosporium and C. necator were varied as follows:Condition M. trichosporium C. necator1 100% (0.2 OD / mL)2 75% (0.15 OD / mL) 25% (0.05 OD / mL)3 50% (0.1 OD / mL) 50% (0.1 OD / mL)4 25% (0.05 OD / mL) 75% (0.15 OD / mL)5 100% (0.2 OD / mL)As shown in Figure 6, the co-cultures exhibited enhanced growth of C. necator in the presence of M. trichosporium. The CFU count of C. necator increased significantly under mixed inoculation conditions (Conditions 2 and 3) compared to its monoculture (Condition 5). This enhancement was most pronounced when AT. trichosporium constituted 50-75% of the initial inoculum.These results establish that co-cultivation of M. trichosporium and C. necator under methane and carbon dioxide conditions enables a metabolically cooperative system. M. trichosporium provides reduced carbon intermediates or organic acids derived from CO2 sequestration, while C. necator assimilates these compounds as well as assimilates CO2 to support enhanced growth. The coculture system thus facilitates integrated carbon recycling and improved biomass yield under mixed-gas cultivation.Additional co-culturing experiments were conducted as follows: a) A co-culture system comprising Methylosinus trichosporium and Cupriavidus necator was established under dual nutrient stress conditions, namely (i) absence of copper (Cu) in the nitrate mineral salts (NMS) medium, and (ii) reduced partial pressure of oxygen (pCL) during fermentation, in the presence of methane (CH4) and carbon dioxide (CO2). The coculture exhibited enhanced CO2 sequestration efficiency as evidenced by increased secretion of organic acids and elevated intracellular accumulation of polyhydroxybutyrate (PHB). b) A co-culture system of M. trichosporium and C. necator was established under nutrientlimited conditions and in the presence of CH4 and CO2. The CO2 sequestration was concentration-dependent, showing a proportional increase in the amounts of secretedorganic acids (e.g., succinate, acetate, formate) and in the intracellular concentration of PHB with increasing relative abundance of M. trichosporium. c) A co-culture system of genetically engineered M. trichosporium (of Example 2) and C. necator was established under nutrient-limited conditions and in the presence of CH4 and CO2. It was seen that the genetically engineered M. trichosporium exhibited increased secretion of organic acids and other reduced carbon intermediates, thereby enhancing C. necator growth and PHB biosynthesis relative to co-cultures containing wild-type M. trichosporium.It will be appreciated that the methods and genetic modifications described herein are not limited to Methylosinus trichosporium but are equally applicable to other methanotrophic and / or mixotrophic bacteria that utilize methane, methanol, or related Cl substrates as carbon and / or energy sources. Comparable results may be obtained using methanotrophs, including but not limited to species of Methylococcus, Methylobacterium, Methylomicrobium, Methylocystis, Methylomonas, Methylocella, and Methylocapsa, or their functional equivalents. Likewise, mixotrophic organisms capable of co-assimilating inorganic (CO2) and organic carbon sources are expected to exhibit analogous phenotypic and metabolic outcomes when subjected to similar genetic modifications or cultivation conditions. Accordingly, the embodiments disclosed herein are representative and not restrictive, and persons skilled in the art will recognize that equivalent strains and related species may be used to achieve substantially similar results without departing from the scope of the present disclosure.Thus, the present disclosure provides efficient methods and systems for the effective and simultaneous utilization of carbon dioxide (CO2) and methane (CH4) using wild type and / or genetically engineered or recombinant methanotrophic microorganisms. Through expression of carbon fixation and redox-balancing modules, methanotrophic microbes are enabled to convert CO2 into reduced carbon intermediates such as formate and organic acids under nutrient-limited or stress conditions. The disclosure further demonstrates synergistic bioprocess configurations or systems wherein metabolites secreted by methanotrophs are utilized by secondary organisms such as autotrophs or heterotrophs to enhance biomass formation and carbon assimilation. Collectively, these approaches establish integrated and efficient methods for the conversion of greenhouse gasesinto value-added compounds / products, thereby providing sustainable solutions for carbon recycling.ADVANTAGES OF THE PRESENT DISCLOSURE

[0187] The present disclosure provides a simple and efficient method and system of converting greenhouse gases (CO2 and CH4) into value-added products. The method can easily be scaled up at an industrial level.

[0188] Since the present method does not employ eukaryote such as algae, the method faces no challenges associated with employing algae such as providing sufficient light intensity at high cell densities, proper agitation, removal of oxygen, nutrient supply etc.The foregoing description of the specific embodiments reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.Throughout this specification, the term ‘combinations thereof or ‘any combination thereof or ‘any combinations thereof are used interchangeably and are intended to have the same meaning, as regularly known in the field of patents disclosures.As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of some embodiments 1 reciting 3 alternatives A, B and C, some embodiments 2 reciting 3 alternatives D, E and F and some embodiments 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D,G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.Any discussion of documents, acts, materials, devices, articles, and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

Claims

WE CLAIM:

1. A method of utilizing methane (CH4) and carbon dioxide (CO2) to produce value-added product, comprising: culturing a methanotrophic bacteria in presence of the methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and culturing the methanotrophic bacteria and optionally a second microbe in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce the value-added product.

2. The method as claimed in claim 1 , wherein the methanotrophic bacteria is a wild-type or recombinant methanotrophic bacteria,3. The method as claimed in claim 1, wherein the methanotrophic bacteria is present in a first bioreactor.

4. The method as claimed in any one of the claims 1 to 3, wherein the second microbe is present in the first bioreactor or a second bioreactor.

5. The method as claimed in any one of the claims 1 -4, wherein the second microbe is a wildtype or a genetically modified bacteria; and wherein the second microbe is an autotroph bacteria, a mixotroph bacteria, or a hydrogenotrophic bacteria.

6. The method as claimed in any one of the claims 1 to 5, wherein the stress is nutrient stress comprising limited copper (Cu) supply, limited oxygen (O2) supply, limited nitrogen (N) supply, limited phosphorus (P) supply, limited carbon (C) supply, limited iron (Fe) supply, limited sulfur (S) supply, limited magnesium (Mg) supply, limited potassium (K) supply, or combinations thereof.

7. The method as claimed in any one of the claims 1-6, wherein hydrogen (H2) is additionally supplied through an external source.

8. The method as claimed in any one of the claims 1-7, wherein the method is carried out at a temperature of about 5°C to 50°C and a pH of about 3 to 8.

9. The method as claimed in any one of the claims 1-8, wherein the first bioreactor or the second bioreactor are selected from a group comprising a stirred tank reactor, an airlift reactor, a bubble column reactor, or a plug flow reactor.

10. The method as claimed in any one of the claims 1-9, wherein the culturing is carried out by batch fermentation, fed-batch fermentation, or continuous fermentation.

11. The method as claimed in claim 1 , wherein the value-added product comprise biomass (rich in lipids, proteins, nucleic acids), organic acids, single cell protein, antibacterial and anticancer drugs, amino acids, vitamins, industrial chemicals, industrial enzymes, biofuel, proteins, peptides, sugars, carbohydrates, fats, fat derivatives (acids, alcohols, acyl CoA, etc.), oils, pigments, secondary metabolites (including carotenoids, terpenoids and others), or combinations thereof.

12. The method as claimed in any one of claims 1 to 11, wherein the methanotrophic bacteria is selected from a group comprising Methylococcus spp., Methylobacterium spp., Methylomicrobium spp., Methylotuvimicrobium spp., Methylocapsa spp., Methylocella spp., Methylosinus spp., Methylobacillus spp., Methylibium spp., Methylacidiphilum spp., Methylophilus spp., Methylomonas spp., Methylovulum spp., Methylomarinum spp., Mycolicibacterium spp., Acidithiobacillus spp., Crenothrix spp., Clonothrix spp., Methylocystis spp., Methylocucumis spp., Methylogaea spp., Methylosarcina spp., Methylosoma spp., and combinations thereof; and wherein the second microbe is selected from a group comprising Cupriavidus spp., Escherichia spp., Aquifex spp., Clostridium spp., Corynebacterium spp., Gordonia spp., Nocardia spp., Rhodobacter spp., Rhodopseudomonas spp., Rhodospirillum spp., Rhodococcus spp., Rhizobium spp., Thiocapsa spp., Pseudomonas spp., Hydrogenomonas spp., Hydrogenobacter spp., Hydrogenophilus spp., Hydrogenovibrio spp., Hydrogenothermus spp., Helicobacter spp., Xanthobacter spp., Hydrogenophaga spp., Bradyrhizobium spp., Ralstonia spp., Alcaligenes spp., Amycolata spp., Aquaspirillum spp., Arthrobacter spp., Azospirillum spp., Variovorax spp., Acidovorax spp., Bacillus spp., Calderobacterium spp., Derxia spp., Flavobacterium spp., Microcyclus spp., Mycobacterium spp., Paracoccus spp., Persephonella spp., Renobacter spp., Streptomyces spp., Thermocrinis spp., Wautersia spp., and combinations thereof.

13. A recombinant methanotrophic comprising at least one gene modification selected from a group comprising: i. overexpressed homologous or heterologous bacteriohemerythrin gene;ii. overexpressed homologous or heterologous genes or pathways for NAD(P)H synthesis or recycling; iii. overexpressed genes encoding one or more enzymes selected from a group comprising carboxylation systems (including carboxylases and carbonic anhydrases), central carbon metabolism, redox balance, and regeneration systems. iv. knocked out native and a heterologous formate dehydrogenase (FDH) genes; or combinations thereof.

14. The recombinant methanotrophic bacteria as claimed in claim 13, wherein the gene is selected from ccfdh (formate dehydrogenase H), fdhD (formate dehydrogenase accessory protein), fits (formate-tetrahydrofolate ligase), zwf (glucose-6-phosphate dehydrogenase), ppc (phosphoenolpyruvate carboxylase), pgi (phosphoglucose isomerase), bhr (bacteriohemerythrin), hoxFUYHWI gene cluster, or combinations thereof.

15. The recombinant methanotrophic bacteria as claimed in any one of the claims 13 or 14, wherein the gene is selected from ccfdh gene having a nucleotide sequence as set forth in SEQ ID NO: 1, or a nucleotide sequence having at least 80% identity to SEQ ID NO. 1;- fdhD gene having a nucleotide sequence as set forth in SEQ ID NO: 2, or a nucleotide sequence having at least 80% identity to SEQ ID NO. 2;- fhs gene having a nucleotide sequence as set forth in SEQ ID NO: 3, or a nucleotide sequence having at least 80% identity to SEQ ID NO. 3; zw / gene having a nucleotide sequence as set forth in SEQ ID NO: 4, or a nucleotide sequence having at least 80% identity to SEQ ID NO. 4;- ppc gene having a nucleotide sequence as set forth in SEQ ID NO: 5, or a nucleotide sequence having at least 80% identity to SEQ ID NO. 5;- pgi gene having a nucleotide sequence as set forth in SEQ ID NO: 6, or a nucleotide sequence having at least 80% identity to SEQ ID NO. 6; bhr gene having a nucleotide sequence as set forth in SEQ ID NO: 7, or a nucleotide sequence having at least 80% identity to SEQ ID NO. 7; and hoxFUYHWI gene cluster, wherein the hoxF, hoxU, hoxY, hoxH, hoxW, and hoxl genes have nucleotide sequences as set forth in SEQ ID NOs: 8 to 13, or nucleotidesequences having at least 80% identity to SEQ ID Nos: 8 to 13; or any combination thereof.

16. The recombinant methanotrophic bacteria as claimed in any one of claim 13 to 15, wherein the gene modification is introduced with a vector comprising one or more expression cassettes encoding the gene as defined in any one of claims 13 or 14.

17. The recombinant methanotrophic bacteria as claimed in any one of the claims 13 to 16 , wherein the vector comprises one or more expression cassettes encoding genes selected from ccfdh,fdhD,jhs, zwf ppc,pgi, bhr, hoxFUYHWIor combinations thereof.

18. The recombinant methanotrophic bacteria as claimed in any one of the claims 13 to 17, wherein the methanotrophic bacteria is selected from a from a group comprising Methylococcus spp., Methylomonas spp., Methylomicrobium spp., Halomonas spp., Methylosarcina spp., Methylocaldum spp., Methylogaea spp., Methylosoma spp., Methyloparacoccus spp., Methyloglobulus spp., Methyloprofundus spp., Methylomarinum spp., Methylovulum spp., Methylomagnum spp., Methylosphaera spp., Methylohalobius spp., Methylothermus spp., Methylomarinovum spp., Acidithiobacillus spp., Crenothrix spp., Clonothrix spp., Methylocystis spp., Methylocucumis spp., Methyloferula spp., Methylosinus spp., Methylocella spp., Methylocapsa spp., Methylacidiphilum spp., Methylacidimicrobium spp., Methylomirabilis spp., Methanoperedens spp., Mycolicibacterium spp. and combinations thereof;.

19. A system for utilizing methane (CH4) and carbon dioxide (CO2), comprising:- a first bioreactor having a methanotrophic bacteria;- a second bioreactor comprising a second microbe; wherein said first bioreactor comprises a first tube coupled to the body of the first bioreactor configured to supply CH4; wherein said first bioreactor comprises a second tube coupled to the body of the first bioreactor configured to supply CO2; wherein the first bioreactor and the second bioreactor are attached to each other by a third tube configured to supply organic acid and H2 from the first bioreactor to the second bioreactor; and wherein said system utilizes the CH4 and CO2 by performing the method as claimed in claims 1 to 12, wherein the method comprises the steps of:- culturing a methanotrophic bacteria in the first bioreactor in presence of methane (CH4), and subjecting the methanotrophic bacteria to stress to produce organic acids and hydrogen (H2); and- culturing the methanotrophic bacteria in the first bioreactor and a second microbe in a second bioreactor in presence of carbon dioxide (CO2), and at least one of the hydrogen and the organic acid to produce the value-added product; wherein the organic acid and hydrogen from the first bioreactor is introduced into the second bioreactor and utilized by the second microbe to produce the value- added product.

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