NOVEL METHANOTHERMOBACTER WOLFEII STRAIN AND USE IN PRODUCTION OF RENEWABLE NATURAL GAS AND CO2 SEQUESTRATION
20260049340 ยท 2026-02-19
Inventors
Cpc classification
C12N1/00
CHEMISTRY; METALLURGY
C12R2001/00
CHEMISTRY; METALLURGY
Y02E50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
The present disclosure pertains to a Methanothermobacter wolfeii archaeal strain identified as BSEL1 and progeny and mutants thereof. The archaeal strain BSEL1 is capable of growing and producing renewable natural gas (RNG) from a supply of carbon dioxide (CO.sub.2) and hydrogen (H.sub.2) without the addition of organic supplements, and further to grow in wastewater with an exceptionally high growth rate. The strain is further capable of reducing CO.sub.2 to a very low level when grown, e.g., in a trickling bed reactor. BSEL1 is of major importance for direct addition of RNG into the natural gas grid.
Claims
1. An engineered strain of Methanothermobacter wolfeii deposited at the American Type Culture Collection under the number PTA-127814, and progeny and mutants thereof.
2. An M. wolfeii culture, comprising the engineered strain of claim 1 and an anaerobic culture medium.
3. The M. wolfeii culture of claim 3, wherein the culture medium is selected from at least one of a wastewater and a mineral media.
4. The M. wolfeii culture of claim 3, further comprising a supply of carbon dioxide gas and a supply of hydrogen gas.
5. The M. wolfeii culture claim 3, wherein the culture medium does not include organic macronutrients.
6. The M. wolfeii culture of claim 3, wherein the culture is maintained at a pH of 8 to 8.5.
7. The M. wolfeii culture of claim 3, wherein the culture is maintained at a temperature of 62-66 C.
8. The M. wolfeii culture of claim 3, further comprising resazurin as a redox indicator and cysteine and/or Na.sub.2S as a reducing agent(s).
9. The M. wolfeii culture of claim 3, wherein the culture does not comprise tungsten.
10. A method of biogas production, comprising: culturing an engineered strain of claim 1 with a supply of carbon dioxide gas and a supply of hydrogen gas under anaerobic conditions.
11. A method of upgrading biogas to renewable natural gas, comprising: culturing an engineered strain of claim 1 with a supply of carbon dioxide gas and a supply of hydrogen gas under anaerobic conditions.
12. A method of carbon sequestration, comprising: culturing an engineered strain of claim 1 with a supply of carbon dioxide gas and a supply of hydrogen gas under anaerobic conditions.
13. The method of claim 13, wherein the carbon dioxide gas is or comprises waste CO.sub.2.
14. A method of producing methane, comprising: culturing an engineered strain of claim 1 in wastewater with a supply of carbon dioxide gas and a supply of hydrogen gas under anaerobic conditions; and recovering methane produced from the culturing step.
15. A culture medium comprising means for converting CO.sub.2 and H.sub.2 to methane in a minimal media, wherein the means is the engineered strain of claim 1.
16. The culture medium of claim 16, which does not comprise tungsten.
17. An engineered strain of Methanothermobacter wolfeii having a formylmethanofuran dehydrogenase F subunit (fwdF) with serine, aspartic acid and isoleucine at positions 17, 69, and 228, respectively, and a D subunit (fwdD) with valine at position 46.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0029] The present disclosure thus describes the isolation and genetic modification of a new variant of Methanothermobacter wolfeii, M. wolfeii BSEL1, an archaeon that utilizes CO.sub.2 as an electron acceptor to produce methane (CH.sub.4) and water (H.sub.2O). The new variant is advantageously capable of minimal nutrient utilization and yet high CH.sub.4 production when cultivated anaerobically in a minimal media such as mineral media, thereby providing a cost-effective methanogen for CO.sub.2 capture.
[0030] Initially, a wild type variant of M. wolfeii was enriched from an anaerobic digester using anaerobic techniques. The original wild type was then genetically modified to produce a mutant capable of growing without additional organic nutritional supplements prior to full isolation. In order to do so, two biological mechanisms were exploited: 1) adaptive laboratory evolution and 2) horizontal gene transfer. Adaptive laboratory evolution selection involves the identification of useful random mutations that take place when cultivating a microorganism repeatedly under the desired conditions, e.g. while diluting the original culture. The mutant recovered after sequential dilutions has higher fitness toward growth in mineral media without supplements. Horizontal gene transfer from other microbes that were present in the microbiome while in culture may also have occurred during the adaptive laboratory evolution procedure. Horizontal gene transfer likely happened prior to full isolation of the mutant strain BSEL1. The methanogen mutant BSEL1 displayed enhanced capabilities to grow at a rapid rate in mineral medium with no organic nutrient addition, as well as in no cost municipal wastewater from a full-scale wastewater treatment facility. BSEL1 grew without organic supplements and surprisingly showed the same growth rate with and without organic supplements. Furthermore, the BSEL1 mutant displayed a high conversion rate of H.sub.2 and CO.sub.2 into CH.sub.4 during different cultivation methods including batch, fed-batch and continuous, as well as several reactor configurations including Balch tubes, bubble-column reactor, and trickling bed reactor.
[0031] In exemplary aspects, the microorganisms of the disclosure are autotrophic. As used herein, the term autotrophic refers to a microorganism capable of using carbon dioxide, formic acid, and/or carbon monoxide as carbon source, and a source of reducing power to provide all carbon and energy necessary for growth and maintenance of the cell (e.g., microorganism). Suitable sources of reducing power may include but are not limited to hydrogen, formate, acetate, and carbon monoxide or electrodes (where they used the electric current) or a combination thereof. In exemplary aspects, the autotrophic microorganisms of the disclosure obtain reducing power from hydrogen.
[0032] In exemplary embodiments, the isolated autotrophic Methanothermobacter microorganism of the disclosure is (a) a microorganism of Methanothermobacter wolfeii strain BSEL, deposited on 16 Aug. 2024, with the American Type Culture Collection (ATCC) under ATCC Patent Deposit Designation No. PTA-127814, (b) a variant of this strain, or (c) a progeny of this strain, wherein the variant or progeny retains, at least in part, the CO.sub.2 conversion phenotypic characteristics of the parent strain M. wolfeii BSEL1.
[0033] M. wolfeii BSEL1 is referred to herein as BSEL1 or BSEL. The 16S rRNA gene sequence and the whole genome of Methanothermobacter wolfeii BSEL1 have been submitted to NCBI GenBank database under the accession numbers OR552920 for the 16S rRNA gene sequence and JAXUHJ000000000 for the whole genome.
[0034] The disclosure also provides a substantially pure culture or monoculture comprising any of the microorganisms of the disclosure and/or is a substantially pure culture. As used herein the term monoculture refers to a population of microorganisms derived or descended from a single species (which may encompass multiple strains) or a single strain of microorganism. The monoculture in some aspects is pure, i.e., nearly homogeneous, except for (a) naturally-occurring mutations that may occur in progeny and (b) natural contamination by non-methanogenic microorganisms resulting from exposure to non-sterile conditions. Organisms in monocultures can be grown, selected, adapted, manipulated, modified, mutated, or transformed, e.g. by selection or adaptation under specific conditions, irradiation, or recombinant DNA techniques, without losing their monoculture nature.
[0035] As used herein, a substantially pure culture refers to a culture that substantially lacks microorganisms other than the desired species or strain(s) of microorganism. In other words, a substantially pure culture of a strain of microorganism is substantially free of other contaminants, which can include microbial contaminants (e.g., organisms of different species or strain). In some embodiments, the substantially-pure culture is a culture in which greater than or about 70%, greater than or about 75%, greater than or about 80%, greater than or about 85%, greater than or about 90%, greater than or about 91%, greater than or about 92%, greater than or about 93%, greater than or about 94%, greater than or about 95%, greater than or about 96%, greater than or about 97%, greater than or about 98%, greater than or about 99% of the total population of the microorganisms of the culture is a single, species or strain of methanogenic microorganism. By way of example, in some embodiments, the substantially pure culture is a culture in which greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total population of microorganisms of the culture is a single methanogenic microorganism species, as disclosed herein.
[0036] In any of the exemplary embodiments described herein, the microorganism may be isolated. As used herein, the term isolated means having been removed from its natural environment, not naturally occurring, and/or substantially purified from contaminants that are naturally associated with the microorganism.
[0037] In other embodiments, the culture comprises a plurality of (e.g., a mixture or combination of two or more) different species of methanogenic microorganisms, one of which is BSEL1. In some aspects, the culture comprises two, three, four, five, six, seven, eight, nine, ten, or more different species of methanogenic microorganisms, one of which is BSEL1. In some aspects, the culture comprises a plurality of different species of methanogenic microorganisms, one of which is BSEL1, but the culture is substantially free of any non-methanogenic microorganism.
[0038] In yet other embodiments, the culture comprises a plurality of microorganisms of different species, in which at least one is a methanogenic microorganism of the present disclosure. In some aspects of this embodiment, the culture comprises BSEL1 and further comprises at least one selected non-methanogenic microorganism. In some aspects, the culture comprises two or more different species of methanogens, of which one is BSEL1, and, optionally comprises at least one selected non-methanogenic microorganism.
[0039] In exemplary aspects, the substantially pure culture or monoculture comprises (a) BSEL1; (b) a variant of BSEL1, or (c) a progeny of BSEL1, wherein the variant or progeny retains, at least in part, the CO.sub.2 conversion phenotypic characteristics of the parent strain BSEL1. Also encompassed are the organism in various states such as dormancy, in which the disclosed microorganisms are not producing methane; an operating or active state in which the disclosed microorganisms are producing methane; active or logarithmic growth phase in which the methanogenic microorganisms are actively multiplying at a rapid rate; stationary phase in which the disclosed microorganisms (after the logarithmic or active growth phase), the rate of cell division and the rate of cell death are in equilibrium or near equilibrium, and thus a relatively constant concentration of microorganisms is maintained in the reactor. etc. All such stages are encompassed.
[0040] As used herein, the term progeny refers to any microorganism resulting from the reproduction or multiplication of M. wolfeii BSEL1, i.e., any descendant of BSEL1. In exemplary embodiments, the progeny are genetically identical to BSEL1, and, as such, the progeny may be considered as a clone of BSEL1. In alternative exemplary embodiments, the progeny are substantially genetically identical to BSEL1, such that the sequence of the genome of the progeny is different from the genome sequence of the microorganism BSEL1 (e.g. exhibiting at least about 95% identity, such as about 95, 96, 97, 98 or 99% identity), but the phenotype of the progeny is substantially the same as the phenotype of BSEL1. In exemplary embodiments, the progeny result from culturing the BSEL1 under the conditions set forth herein for the parent BSEL1. In other words, the variant retains the CO.sub.2 conversion phenotypic characteristics of the microorganism of BSEL1, as described herein.
[0041] Other forms of BSEL1 include those intended to preserve the archaea for lengthy periods of time, for transportation and shipping, etc. These forms include but are not limited to desiccated and/or freeze-dried forms from which most (at least 75, 80, 85, 90, 95% or more) of the water has been removed; various concentrates of the microorganism; etc. Such forms encompassed the archaea e.g. in the form of granules, pellets, powders, etc.
Phenotypic Characteristics
[0042] The phenotypic characteristics of interest that are present in BSEL1 and variants and progeny thereof include but are not limited to:
[0043] i) The ability to grow and produce CH.sub.4 from CO.sub.2 and H.sub.2 in media that is devoid of a dedicated supply of organic macro- and micronutrients, e.g. BSEL1 grows at maximum rate in minimal media, mineral media, wastewater, wastewater with added vitamins, tap water with added vitamins, etc. Under these minimal conditions, the microorganism produces CH.sub.4 at a rate of at least about 66 mL standard temperature and pressure (0 C. and 1 atm) per hour (STP h.sup.1) and up to 434 mL STP h.sup.1 (e.g. about 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 or 12.0 mL STP h.sup.1) in systems with less than 4 L of reaction volume without optimized gas-liquid mass transfer. In systems with optimized gas-liquid mass transport, BSEL1 supports a high gas rate and produces CH.sub.4 at a rate of at least 435 mL STP h.sup.1 and up to 969 mL STP h.sup.1.
[0044] ii) M. wolfeii BSEL1 grew at pHs between 6.5 and 8.6, with a maximum growth rate of 0.270.03 h.sup.1 at pH 8.3.
[0045] iii) The growth temperature range of the BSEL1 strain was between 45 C. and 75 C., with optimum growth at 64 C.
[0046] iv) BSEL1 growth is non-dependent on tungsten, in contrast to other M. wolfeii strains having metalloenzymes that depend on this metal as a cofactor.
[0047] v) Unlike other methanogenic archaea, M. wolfeii BSEL1 stains Gram negative.
[0048] vi) The F subunit (fwdF) of M. wolfeii BSEL1 formylmethanofuran dehydrogenase has the amino acids serine, aspartic acid and isoleucine at positions 17, 69, and 228, respectively, and the D subunit (fwdD) has valine at position 46.
[0049] In exemplary embodiments, the variant exhibits or comprises certain characteristics or features, which, optionally, may be specific to a given growth phase (active growth phase, stationary growth phase, nearly stationary growth phase) or state (e.g., dormant state, operating state). For example, in some embodiments, the variant microorganism has been modified to survive and/or grow in a desired culture condition which is different from a prior culture condition in which BSEL1 survived and/or grew. The desired culture conditions may differ from the prior environment in temperature, pH, pressure, cell density, volume, humidity, salt content, conductivity, carbon content, nitrogen content, vitamin-content, amino acid content, mineral-content, or a combination thereof. In some embodiments, the methanogenic microorganism, before adaptation in culture or genetic modification, is one that is not a halophile but, through adaptation in culture or genetic modification, has become a halophile. As used herein, halophile or halophilic refers to a microorganism that survives and grows in a medium comprising a salt concentration higher than 100 g/L.
Culture Medium
[0050] A culture comprising the BSEL1 methanogenic microorganisms, may be maintained in or on a culture medium. In some embodiments, the culture medium is a solution or suspension (e.g., an aqueous solution). In other embodiments, the culture medium is a solid or semisolid. In yet other embodiments, the culture medium comprises or is a gel, a gelatin, or a paste.
[0051] In some embodiments, the culture medium is one that encourages the active growth phase of the methanogenic microorganisms. In exemplary aspects, the culture medium comprises materials, e.g., nutrients, in non-limiting amounts that support the various phases or states of BSEL1, such as a relatively high exponential growth phase of BSEL1. The materials and amounts of each material of the culture medium that supports the active phase of the methanogenic microorganisms typically vary depending on the species or strain of a microorganism. However, it is within the skill of the ordinary artisan to determine the contents of culture medium suitable for supporting the active phase of the microorganisms of the culture. In some embodiments, a culture medium supports the exponential growth. Exemplary components of media for BSEL1 include but are not limited to: Inorganic Materials: Inorganic Elements, Minerals, and Salts
[0052] In some embodiments, the medium for culturing archaea comprises one or more nutrients that are inorganic elements, or salts thereof. Common inorganic elements include but are not limited to sodium, potassium, magnesium, calcium, iron, chloride, sulfur sources such as hydrogen sulfide or elemental sulfur, phosphorus sources such as phosphate and nitrogen sources such as ammonium, nitrogen gas or nitrate. Exemplary sources include NaCl, NaHCO.sub.3, KCl, MgCl.sub.2, MgSO.sub.4, CaCl.sub.2, FeSO.sub.4, Na.sub.2HPO.sub.4, NaH.sub.2PO.sub.4, H.sub.2S, Na.sub.2S, NH.sub.4OH, N.sub.2, and NaNO.sub.3. In some embodiments, the culture medium further comprises one or more trace elements selected from the group consisting of ions of barium, bromine, boron, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, tungsten and aluminum. These ions may be provided, for example, in trace element salts,
[0053] In some embodiments, the medium comprises one or more minerals selected from the group consisting of nickel, cobalt, sodium, magnesium, iron, copper, manganese, zinc, boron, phosphorus, sulfur, nitrogen, selenium, optionally tungsten, aluminum and potassium including any suitable non-toxic salts thereof. Thus, in some embodiments, the minerals in the medium are provided as mineral salts. Any suitable salts or hydrates may be used to make the medium. In some embodiments, L-cysteine may be added as a reduction-oxidation (redox) buffer to support early phases of growth of a low-density culture.
[0054] In some embodiments, the media comprises a nitrogen source, e.g., ammonium hydroxide or ammonium chloride in an amount of about 1 mM to about 10 mM, e.g. 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or any combination of the foregoing range endpoints.
[0055] In some embodiments, the microorganism is adapted to prefer high salt conditions, e.g. about 1.5 M to about 5.5 M NaCl, or about 3 M to about 4 M NaCl. In some embodiments, the microorganism is adapted to growth in higher salt conditions than their normal environment.
[0056] Conditions for culturing the microorganisms disclosed herein may vary, depending, e.g. on the source of CO.sub.2, the location of the culture, etc. For example, for laboratory and/or industrial CH.sub.4 production, the culture medium is generally and advantageously, a BA medium comprising the following components expressed as [mg/L]: NH.sub.4Cl [1000]; NaCl [100]; NaHCO.sub.3[2600], MgCl.sub.2*6H.sub.2O [100]; CaCl.sub.2)*2H.sub.2O [50]; K.sub.2HPO.sub.4*3H.sub.2O [400]; H.sub.3BO.sub.3 [0.1]; ZnCl.sub.2 [0.1]; CuCl.sub.2*2H.sub.2O [0.04]; MnCl.sub.2*2H.sub.2O [0.04]; (NH.sub.4).sub.2MoO.sub.4*4H.sub.2O [0.1]; AlCl.sub.3*6H.sub.2O [0.1]; CoCl.sub.2*6H.sub.2O [0.1]; NiCl.sub.2*6H.sub.2O [0.1]; ethylenediaminetetraacetic acid Na-salt (EDTA) [0.5]; Na.sub.2SeO.sub.3*5H.sub.2O [0.1]; FeCl.sub.2*4H.sub.2O [2]; biotin [0.02]; folic acid [0.02]; pyridoxine hydrochloride [0.1]; riboflavin [0.05]; thiamine hydrochloride [0.05]; cyanocobalamin [0.001]; nicotinic acid [0.05]; p-aminobenzoic acid [0.05]; lipoic acid [0.05]; DL-pantothenic acid [0.05]; and resazurin sodium salt [0.5].
[0057] Those of skill in the art will recognize that the precise type of component and the amount of each can vary and are best determined by small-scale testing. For example, other possible components include but are not limited to one or more of: CaSO.sub.4; H.sub.3BO.sub.3; Co(NO.sub.3).sub.2; and CuSO.sub.4, and various salts and hydrates thereof. The formula may be adjusted, and/or standardized to suit desired performance parameters.
[0058] In some exemplary aspects, BSEL1 is cultured in a minimal media (e.g. for example, tap water) with added vitamins. Vitamins which are added under these growth conditions may include but are not limited to: ascorbic acid, biotin, choline chloride; pantothenate, folic acid, i-inositol, menadione, niacinamide, nicotinic acid, para-aminobenzoic acid (PABA), pyridoxal, pyridoxine, riboflavin, thiamine-HCl, vitamin A acetate, vitamin B12 and vitamin D2. In some embodiments, the medium is supplemented with a vitamin that is essential to survival of the methanogenic microorganism, but other vitamins are substantially absent. In some aspects, the vitamins include but are not limited to riboflavin; thiamine; cyanocobalamin; one or more B-complex vitamins such as nicotinic acid (vitamin B3), and the like.
[0059] Typically, the culture medium requires a source of H.sub.2. Hydrogen gas may be produced from a variety of sources. In one embodiment, inexpensive electric power can be used to produce hydrogen from water via electrolysis. For example, an integrated electrolysis/methane fermentation system can be viewed as converting an intermittent energy source (e.g. inexpensive off-peak electricity from power plants) to a stable chemical energy store, using hydrogen as an intermediate and methane as the final energy carrier. Other sources include the thermal breakdown of water, water radiolysis, steam reforming of natural gas, biogenic H.sub.2 produced by acidogenic bacteria, and the like.
[0060] Further, in some aspects, the culture medium is or comprises what would otherwise be waste CO.sub.2, i.e., a waste stream, such as waste water, partially treated waste water, sewage, sewage sludge, sewage sludge digester gas, contaminated liquids, atmospheric CO.sub.2, CO.sub.2 extracted from a source where it is unwanted and stored underground, combustion flue gases, etc.
[0061] The pH of the culture medium generally ranges from about 7.0 to about 9.0, such as about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8 or 9.0. The range is generally from about pH 8.0 to about 8.6, such as about 8.3.
[0062] The temperature of cultivation is generally from about 55 to about 75, such as about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. The temperature range is generally from about 60 to about 70, such as about 64.
[0063] Other possible media components and culture conditions for methanogens in general are described in detail, for example, in published United States patent application US20220170050, the complete contents of which is hereby incorporated by reference in entirety.
[0064] In some embodiments, components required for the growth of non-methanogenic organisms are substantially absent from the media. Such components include, but are not limited to, one or more organic carbon sources, and/or one or more organic nitrogen sources, and/or one or more vitamins and/or tungsten. In some embodiments, formate, acetate, ethanol, methanol, methylamine, and any other metabolically available organic materials are substantially absent from the media.
[0065] In some aspects, tungsten is absent from the media.
[0066] In some embodiments, the cultures are maintained in a culture vessel within a pressure range from about 0.5 atmospheres to about 500 atmospheres. The culture can be maintained with a source of intermittent agitation, shaking, stirring, and the like. Also in exemplary embodiments, the methane gas removed from the culture suitably comprises less than about 450 ppm hydrogen sulfide, or alternatively less than about 400 ppm, 300 ppm, 200 ppm, 150 ppm, 100 ppm, 50 ppm or 20 ppm of hydrogen sulfide. Total gas delivery rates (CO.sub.2) in the range of 0.2 to 4 volume of gas (STP) per volume of culture per minute are suitable, since they both maintain and exploit high volumetric concentrations of active methane generation capacity.
Exemplary Systems
[0067] The disclosure furthermore provides systems and/or apparatuses and methods for converting carbon dioxide into methane. The systems include, and the methods employ, a supply of carbon dioxide, a source of reducing power, a microorganism in accordance with the disclosure or a variant or progeny thereof, as described herein, within a biological reactor. A biological reactor, also known as a fermentor vessel, bioreactor, or simply reactor, as set forth herein may be any suitable vessel in which methanogenesis can take place. Suitable biological reactors should be sized relative to the volume of the CO.sub.2 source. Typical streams of 2,200,000 lb CO.sub.2/day from a 100,000,000 gal/yr ethanol plant would require a CO.sub.2 recovery/methane production fermentor of about 750,000 gal total capacity. Fermentor vessels similar to the 750,000 gal individual fermentor units installed in such an ethanol plant would be suitable.
[0068] In exemplary aspects, the source of reducing power (in whole or in part) is hydrogen, e.g., hydrogen (H.sub.2) gas. Accordingly, in exemplary aspects, the system (e.g. a bioreactor) of the present disclosure includes a supply of carbon dioxide, a supply of hydrogen gas, and a microorganism in accordance with the disclosure or a variant or progeny thereof, as described herein. Examples of suitable bioreactors include but are not limited to: stratified bioreactors, cascaded bioreactors, electro-biological apparatuses, e.g. Systems and apparatuses of this category are further described in International Patent Application No. PCT/US2010/040944, filed Jul. 2, 2010, and published United States patent application US20210115477A1, the complete contents of each of which is incorporated herein by reference in its entirety. Generally, the methanogenic microorganisms disclosed herein are cultured, for example, in shake or stirred tank bioreactors, hollow fiber bioreactors, or fluidized bed bioreactors, and operated in a batch, fed batch, continuous, semi-continuous, or perfusion mode.
[0069] In an exemplary method, methane is produced from electric power in a two-step process in a bioreactor. The first step uses electric power to make hydrogen gas from water in a standard water electrolysis system. In a second step, the hydrogen gas is then pumped into a methanogenic reaction chamber, such as a biological reactor as is described in greater detail in U.S. Publ. No. 2009/0130734 which is incorporated in its entirety herein by reference. The reaction chamber comprises a culture of the microorganisms described herein, e.g. within a container that forms part of the bioreactor.
[0070] A bioreactor, also known as a digestor or fermenter vessel, as set forth in this disclosure is any suitable vessel in which methanogenesis can take place. Suitable bioreactors should be sized relative to the volume of the carbon dioxide source balanced with its specific need for hydrogen. Typical streams of 2,200,000 lb carbon dioxide/day from a 100,000,000 gal/yr ethanol plant would require a carbon dioxide recovery/methane production bioreactor of about 750,000 gal total capacity. Vessels similar to the 750,000 gal individual fermentor units typically installed in such an ethanol plant may thus be suitable.
[0071] An exemplary bioreactor system is depicted schematically in
In some aspects, a manufacturing plant, e.g. a fuel methane plant, uses the microorganisms described herein. The input to the plant includes e.g. an industrial CO.sub.2 source from combustion or biological produced CO.sub.2 from conversion of organics. An electrolyzer produces hydrogen, which is also collected and stored before use in the plant. The hydrogen and carbon dioxide from their respective storage tanks are optionally fed through an oxygen scrubber for removal of oxygen from the carbon dioxide. After passing through the oxygen scrubber, the hydrogen and carbon dioxide are fed into a digestor/fermentor/bioreactor system for conversion of carbon dioxide and hydrogen to methane. The methane gas vented from the system optionally passes through a sulfur scrubber for recovering sulfur from the product methane stream. The methane gas can then be stored in a methane storage tank or injected into the natural gas grid.
[0072] Other exemplary systems are described, for example, in published United States patent application US20220170050, the complete contents of which is hereby incorporated by reference in entirety.
Uses
[0073] Gas fermentation with M. wolfeii BSEL1 may be used to convert any or all of various CO.sub.2 waste streams/sources to methane, including but not limited to: industrial sources such as syngas, combustion flue gases, steelmaking slag, municipal solid waste incinerator ashes, combustion fly ash, black liquor, paper mill waste, mining waste, cement waste, construction and demolition waste, waste from the organic industry, flue gas desulfurization gypsum waste, CO.sub.2 generated by manufacturing, mining, agriculture, and energy production, production of ethanol, electricity and heat production, transport, combined cycle coal fired energy plants; during space missions such as its use as a biocatalyst to transform CO.sub.2 from e.g., the Martian atmosphere or from the exhaust breath of astronauts (the resulting CH.sub.4 could be liquified and used as rocket fuel or as an alternative source for electricity generation in microturbines); and the like. M. wolfeii BSEL1 may be used in conjunction with or instead of any carbon capture method, e.g. any point source capture technology or any general capture technology, examples of which include but are not limited to: geologic (underground) storage facilities, pipelines, saline formations, etc.
[0074] Additional examples of processes that that produce CO.sub.2 that can be captured by the present systems and methods include biomass fermentation to produce liquid fuels and coal and biomass gasification processes. Gasification is a process that converts carbonaceous materials, such as coal, petroleum, petroleum coke or biomass (living or dead biological material), into carbon monoxide, hydrogen and carbon dioxide.
Example
Phenotypic and Genomic Characterization of Methanothermobacter wolfeii Strain BSEL1, a CO.sub.2-Capturing Archaeon with Minimal Nutrient Requirements
[0075] This example describes the phenotypic and genomic characterization of a new Methanothermobacter wolfeii variant with evolutionary adaptations that allow the strain to thrive in an environment with H.sub.2 as the sole electron donor and CO.sub.2 as the only carbon source. Isolation began with serial dilution of anaerobic digestate in minimal medium until a culture capable of growing without any organic carbon addition, even at high dilutions of 10.sup.8, was developed. Isolation was performed using modified Hungate techniques (Wolfe RS. 2011. Techniques for cultivating methanogens. Methods Enzymol 494:1-22). The isolated archaeal strain was capable of growing at maximum rates in minimum media with CO.sub.2 as the only carbon source and low levels of NH.sub.4Cl as sole nitrogen source. Prolonged cultivation of the new wolfeii isolate using minimum additions of minerals and vitamins in the absence of yeast extract showed a consistently high growth rate, demonstrating that the new isolate was stable under these conditions. Optimum temperature and pH for growth of the new isolate were determined. In addition, the new M. wolfeii BSEL1 isolate was grown in a continuous gaseous bioreactor to demonstrate the potential of this strain for CO.sub.2 conversion with H.sub.2 into CH.sub.4. The whole genome of the new variant was de novo assembled and annotated. A comparative analysis of orthologous genes in Methanothermobacter wolfeii BSEL1 and the type strain DSM 2970 displayed differences in the abundance of relevant genes associated to energy metabolism, amino acids synthesis, post-translational modifications, and nitrogen fixation.
Adaptation, Isolation, and Identification of the Methanogen
[0076] Methanothermobacter wolfeii BSEL1 was isolated from a thermophilic anaerobic digester via enrichment cultivation (See
[0077] An aliquot from the last generation of the enriched culture was serially diluted and transferred to agar BA medium in roll tubes. The roll tubes were incubated in a pressurized headspace of H.sub.2 and CO.sub.2 (134.31 kPa absolute pressure) at 55 C. for 15 days. At the end of this period, isolated tannish-white colonies growing on the surface and deep inside the agar were observed (see
[0078] The new isolate showed 99% nucleotide sequence identity with the taxon Methanothermobacter wolfeii. As shown in
Optimal Growth Conditions of Methanothermobacter wolfeii Strain BSEL1
[0079] The optimal temperature and pH conditions to cultivate M. wolfeii BSEL1 were determined using optical density (OD590) as the response variable in quantitative growth assays (n=3-5). The specific growth rate () was calculated using the logistic growth model (equation 3) (see Materials and Methods section). The temperature and pH response curves for M. wolfeii BSEL1 are shown in
TABLE-US-00001 TABLE 1 Phenotypic characteristics of Methanothermobacter wolfeii and variants Cell Optimum Optimum Maximum Isolate Source morphology Temp. ( C.) pH reported (h.sup.1) DSM Mix of river Slender rods 65 7.0-7.5 0.17 2970* sediment and in chains sewage sludge (Germany) OCM36** Sewage sludge Slender rods 56 8.0 nr digestate in crooked (China) filaments SIV6*** Sewage sludge nr nr nr nr digestate (Germany) BSEL1 Manure and Slender rods 64 8.3 0.27 0.03 (This sewage in crooked work) sludge digestate filaments (USA) nr = not reported *Winter et al. 1984. Syst Appl Microbiol 5: 457-466. **Zhao et al. 1986. Applied and Environmnetal Microbiol 52: 1227-1229. ***Maus et al. 2016. Biotechnol Biofuels 9: 171.
Effect of Nutrients on the Growth Rate
[0080] To assess the effect of YE and vitamins on M. wolfeii BSEL1, a set of growth assays were performed in the presence of these additives at 55 C. A set of samples without adding YE and vitamins to the medium was used as a control (see
To determine the influence of low concentrations of NH.sub.4Cl as sole nitrogen source on the growth rate of M. wolfeii BSEL1, a growth assay was conducted using various NH.sub.4Cl concentrations ranging from 1.9 to 10.3 mM in BA medium (see
[0081] By analyzing the linearized growth curves from the previous experiment (see
Longevity of Methanothermobacter wolfeii BSEL1 in Semi-Continuous Cultivation
[0082] To assess the longevity of M. wolfeii BSEL1 when cultivated under a minimum supply of nutrients, a semi-continuous 2 L bioreactor was inoculated with the BSEL1 isolate and operated at 55 C. with constant agitation (120 rpm) for 8 months. This reactor was fed with a 70% H2:30% CO.sub.2 mixture (134.31 kPa absolute pressure) every 2-3 days. The gas in the headspace of the bioreactor was replaced every feeding cycle with the 70% H2:30% CO.sub.2 mixture to prevent accumulation of CH.sub.4 in the system. Fresh BA medium was added in aseptic conditions once every week with a dilution rate of 1 mL of fresh medium per milliliter of culture broth. At the end of the 8-month period, the culture displayed continuous methanogenic activity, with >50% v CH.sub.4 in the headspace. Besides, fluorescence microscopy revealed the presence of coenzyme F420 in the cells after 8 months of cultivation and only the M. wolfeii BSEL1 morphotype was present (
[0083] In addition, it was observed that M. wolfeii BSEL1 maintained viability for more than 48 months of cryopreservation in glycerol. Storage at room temperature in BA medium under a headspace of N.sub.2 was also an effective storage method to preserve the microorganism without signs of viability loss for up to 6 months.
CO.SUB.2.-Capture and Methanation Assay in Bubble Column Gas Fermentation
[0084] The capability of M. wolfeii BSEL1 as biocatalyst for CO.sub.2 conversion into CH.sub.4 was tested using an 8 L bubble column gas fermentation bioreactor fed with a gas mixture mimicking the biogas from sewage sludge anaerobic digestion (66.5% CH.sub.4 and 33.5% CO.sub.2). This biogas mimic was blended with H.sub.2 to a H.sub.2:CO.sub.2 ratio of 4, in accordance with the stoichiometry of hydrogenotrophic methanogenesis. The gaseous substrate was held in a pressurized tank and sparged at the bottom of the bioreactor. The headspace was kept at a constant pressure of 134.31 kPa, controlled by a pressure switch, without removing gases from the device (except for sampling). Prior to use, the bioreactor was autoclaved at 120 C. for 1 h. Sterile BA medium was added to the bioreactor in anoxic/aseptic conditions before operation. The reactor was operated at 550.2 C. and the pH was regulated with a bicarbonate buffer contained in the medium. The composition of the headspace in the bioreactor was analyzed over time using mass spectrometry (see
Genome Assembly and Genomic Features of the BSEL1 Strain
[0085] Whole-genome sequencing of M. wolfeii BSEL1 DNA was performed via next-generation sequencing. 5.8 GB of sequencing data were obtained. FastQC was used for a quality check. Low-quality reads were trimmed using Trimmomatic, conserving 45.3 million reads (forward+reverse) with an average Q-score of 34 (lengths of 30-250 bp). The trimmed reads were de novo assembled using two different tools SPAdes and SKESA. The draft assemblies were examined by comparing their completeness and the copy number of key genes that translate into the main metabolic and stress response functions in methanogens (mcr, acs, frh, por, and trx). The draft assemblies were found to be comparable, both displaying a completeness of 90.6% and the same copy number for all the key genes inspected. Ultimately, the genome assembled with SKESA was selected for downstream processing, as SKESA has been reported to generate 13% less mismatches than SPAdes. The final genome of M. wolfeii BSEL1 consisted of 1.66 kbp. The similarity of conserved regions in the whole-genome sequence of the BSEL1 strain with closer relatives from the Methanothermobacter genus was assessed by calculating the average nucleotide identity (ANI). It was confirmed that the closest phylotype to the BSEL1 strain is M. wolfeii DSM 2970 (ANI of 99.35), followed by Methanothermobacter marbur gensis (83.63) and Methanothermobacter defluvii (83.43). The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).
[0086] The chromosomal map of the BSEL1 strain is shown in
TABLE-US-00002 TABLE 2 Annotation details of M. wolfeii BSEL1 and M. wolfeii DSM 2970 genomes through NCBI PGAP M. wolfeii M. wolfeii DSM 2970 Features BSEL1 (type strain) Size (Mbp) 1.66 1.71 Total genes 1,842 1,890 Protein coding sequences 1,796 1,838 (CDS) Hypothetical proteins 1,411 (79% of 1,425 (75% of CDS) CDS) CDS with unidentified 917 (50% of 966 (51% of function (Kyoto CDS) CDS) Encyclopedia of Genes and Genomes) Total non-coding RNA 41 46 genes GC content 48.9% 48.5% Completeness 90.6% 89.6%
[0087] An analysis of orthologous genes was conducted in both the BSEL1 and the type strain by classifying the protein coding genes (coding sequences) of both strains into the functional categories of the Kyoto Encyclopedia of Genes and Genomes (KEGG) as well as the Cluster of Orthologous Genes (COG). This study was tailored to identifying predicted functional features in the BSEL1 strain that may lead to the differences in phenotype with respect to the type stain. The abundances of the various genes/proteins classified in KEGG categories and COG classes are shown in
DISCUSSION
[0088] Methanothermobacter members are ubiquitous in anaerobic environments. Their genomic signature has been identified in numerous habitats such as lake sediments, hot springs, water-flooded oil fields, and mammalian guts, as well as human-shaped ecosystems such as agricultural soils, landfills, and anaerobic digesters. Hydrogenotrophic methanogens occupy approximately 1% of the microbiome of anaerobic digesters. The enrichment cultivation pipeline shown in
[0089] The amino acids sequences of the genes with identity scores lower than 0.5 located in the non-conserved 550-680 kbp region of the BSEL1 strain genome were recognized and queried with BLASTp. Some of the putative proteins encoded in the mentioned region included YkvA family protein, ATP-dependent DNA helicase PcrA, DUF169 and DUF2085 domain-containing proteins, carboxypeptidase regulatory-like domain-containing protein, ABC transporter ATP-binding protein, zinc ribbon domain-containing protein, pseudomurein-binding protein, tetratricopeptide repeat protein, RAD55 family ATPase, along with hypothetical proteins with unknown functions. Most of these proteins were discovered in other members of the same genus such as Methanothermobacter sp. THM-1, Methanothermobacter sp. CaT2, and Methanothermobacter thermautotrophicus, as well as metagenomic material from uncultured archaea from various phyla (Euryarchaeota and Thermoproteota) found in thermophilic anaerobic environments, thus supporting the occurrence of horizontal gene transfer evolutionary events from close neighbors cohabiting the anaerobic digester from where the BSEL1 strain was isolated.
[0090] Morphologically, the BSEL1 strain is similar to other Methanothermobacter members. All of them lack motility, do not have archaeal appendages (such as fimbriae and canulae), and do not generate endospores (see Table 1). On the other hand, relevant physiological differences were found between M. wolfeii BSEL1 and other isolates from the same species. For instance, the maximum specific growth rate reported for the type strain DSM 2970 was 0.17 h.sup.1 in the presence of YE, while the BSEL1 strain grew at 0.27 h.sup.1 at optimal temperature and pH without the addition of organic carbon and nitrogen to the medium (as shown in
[0091] The lack of nutrients during the isolation procedure likely favored the selection of a mutant adapted to grow on CO.sub.2 as the only carbon source with NH.sup.4+ as the nitrogen source. Interestingly, this implies that M. wolfeii BSEL1 can synthesize the necessary precursors for its biosynthesis processes, including proteinogenic amino acids, solely from inorganic molecules. In contrast, the type strain M. wolfeii DSM 2970 and the model organism M. thermautotrophicus were both isolated using media that contained formate as an alternative carbon source and YE, which provided additional organic carbon and nitrogen, including amino acids. Thus, it is unclear if M. wolfeii DSM 2970 and M. thermautotrophicus could synthesize all the necessary building blocks for cell growth without the addition of organic precursors to the medium. The overabundance of homoserine dehydrogenase in the genome of the BSEL1 strain suggests that the type strain may have a truncated pathway for lysine biosynthesis, thus explaining its dependency on the addition of this amino acid for growth. The minimal medium (BA medium) used to isolate and characterize M. wolfeii BSEL1 in this study had lower concentrations of several minerals when compared with the anaerobic medium originally used to cultivate M. wolfeii DSM 2970 by Winter et al. (see Table 3). These minerals with lower content in BA medium were Na.sup.+, Mg.sup.2+, Al.sup.3+, Ca.sup.2+, Mn.sup.2+, Fe.sup.2+, CO.sub.2.sup.+, Ni.sup.2+, Cu.sup.2+, Zn.sup.2+, BO.sup.3, HCO.sup., and MoO.sup.2+. This likely indicates that the BSEL1 isolate requires a relatively low content of minerals for growth. Interestingly, the Mg, Ca, Mn, Fe, Cu, and Zn contents in BA medium are equal or lower to the ionic background found in tap water and some bottled waters in the USA. Accordingly, tap water can be sufficient for cultivating the strain when only small amounts of Na, Al, K, Co, Ni, Mo, Cl, B, Se, NH.sup.+ and vitamins are added. Besides BA medium, all anaerobic media formulations reported in the literature also include resazurin as a redox indicator and cysteine and/or Na.sub.2S as a reducing agent(s). These are unavoidable components to ensure anaerobic conditions.
TABLE-US-00003 TABLE 3 Minimal conditions for Methanothermobacter wolfeii cultivation reported in the literature, in contrast with the present work Organic growth Inorganic factors and/or nitrogen Doubling Isolate Mineral nutrients organic nitrogen source e.sup. Temp Initial time designation [mM] sources [mM] donor ( C.) pH (h) reference NaOH [50], NaHCO.sub.3 [71], NaCl [8.8], Vitamins + NH.sub.4.sup.+ [3.8] H.sub.2 45 7.0 8 DSM CaCl.sub.2 [0.49], K.sub.2HPO.sub.4 [1.5], KH.sub.2PO.sub.4 0.2% YE + 55 4 2970; [1.9], MgSO.sub.4 [0.66], CoCl.sub.2 [0.008], 0.2% peptone 60 4 type FeSO.sub.4 [0.013], MnSO.sub.4 [0.033], 65 4 strain ZnSO.sub.4 [0.006], CuSO.sub.4 [0.001], 72 12 (*) AlKSO.sub.4 [0.0004], Na.sub.2WO.sub.4 [0.0038], H.sub.3BO.sub.3 [0.0016], Na.sub.2MoO.sub.4[0.0005] NaHCO.sub.3 [31], NaCl [19], MgCl.sub.2 Vitamins NH.sub.4.sup.+ [18.7] H.sub.2 45 6.8 14.7 6.1 BSEL1 [0.5], CaCl.sub.2 [0.3], K.sub.2HPO.sub.4 [1.8], Vitamins 50 6.1 1.7 (This CoCl.sub.2 [0.0002], MnCl.sub.2 [0.0003], FeCl.sub.2 10% Vitamins 55 4.3 1.3 work) [0.01], ZnCl.sub.2 [0.0004], AlCl.sub.3 [0.0004], Vitamins 4.5 0.7 CuCl.sub.2 [0.0002], NiCl.sub.2 [0.0004], Vitamins + 10.6 2.0 Na.sub.2SeO.sub.3 [0.0004], H.sub.3BO.sub.3 [0.0008], 0.1% YE (NH.sub.4).sub.2MoO.sub.4 [0.0002] 10% Vitamins + 12.4 2.0 0.1% YE Vitamins 64 3.2 0.1 Vitamins 71 3.7 0.2 Vitamins 73 8.4 0.4 Vitamins 64 8.3 2.5 0.3 Vitamins NH.sub.4.sup.+ [10.3] 55 6.8 5.3 0.5 NH.sub.4.sup.+ [6.1] 5.5 NH.sub.4.sup.+ [2.8] 5.2 NH.sub.4.sup.+ [1.9] 5.4 Wastewater 7.2 5.0 1.2 Wastewater + vitamins 7.2 4.1 0.6 .sup.aRanges () represent confidence intervals from the mean (a = 0.05, n = 3-5). Composition of vitamins in the medium is reported in Materials and Methods. *Winter et al. 1984. Syst Appl Microbiol 5: 457-466.
[0092] Minerals play a vital role in the metabolism of prokaryotic cells. Among other functions, minerals serve as cofactors for metalloenzymes and as signaling molecules in crucial regulatory pathways of methanogenesis. Methanothermobacter wolfeii was originally described by Winter et al. as a tungsten-dependent methanogen. Here, we show that this characteristic is not shared among all variants of the species, as M. wolfeii BSEL1 grew at maximum rates in BA medium, which lacked tungsten (see Table 3). This metal is responsible for the electron transfer in the active site of ferredoxin oxidoreductases and formyl dehydrogenases. These groups of enzymes have main roles in the Wolfe cycle. For instance, formylmethanofuran dehydrogenase (EC 1.2.7.12) catalyzes the CO.sub.2 fixation step of methanogenesis. Formylmethanofuran dehydrogenase is often described as tungsten-dependent. However, Bevers et al. showed that some formylmethanofuran dehydrogenases in anaerobic archaea use molybdenum, which has the same number of valence electrons as tungsten. Interestingly, the amino acids sequences of all the subunits of formylmethanofuran dehydrogenase encoded in M. wolfeii BSEL1 genome present multiple substitutions when compared with the peptide sequences of the type strain. For instance, the subunit F (fwdF) has the substitutions T.fwdarw.S, N.fwdarw.D, and V.fwdarw.I at the positions 17, 69, and 228 of the peptide sequences, respectively, while the subunit D (fwdD) has the substitution A.fwdarw.V at the position 46. These substitutions may be responsible for the allocation of molybdenum in the formylmethanofuran dehydrogenases as well as the tungsten independence of the BSEL1 strain.
[0093] The biosynthesis of amino acids in microorganisms is of conspicuous importance, as it constitutes ca. 20% of the total energy spent during protein synthesis. Methanogens use the Wood-Ljungdahl pathway for CO.sub.2 and H.sub.2 fixation into formate. Later on, formate is transformed into other organic intermediates aided by the central carbohydrate metabolism. Glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway lead the formation of precursors for the synthesis of amino acids. The only amino acids previously known to be synthesized fully autotrophically, directly from CO.sub.2 and NH.sup.+ in M. wolfeii DSM 2970, are arginine and proline. Based on the aforementioned, it is likely that all methanogenic species are capable of synthesizing amino acids solely from inorganic primary substrates; however, as shown by Winter et al., their growth rate without the addition of organic carbon sources is significantly reduced. This is, however, not the case of the BSEL1 strain. The addition of YE to the medium for prokaryotes cultivation is a collective practice in microbiology, including practices to cultivate hydrogenotrophic methanogens. Yet, in this work, we found that M. wolfeii BSEL1 was inhibited even by low concentrations of YE (0.1%) (
[0094] The duplication time of M. wolfeii BSEL1 on wastewater with vitamins added (4.10.6 h) was equivalent to BA medium (4.50.7 h) at the same temperature (see
[0095] The results presented in
[0096] During the first 21 h of cultivation of M. wolfeii BSEL1 in the bubble column bioreactor, it was estimated that ca. 0.9 mmol CO.sub.2 L-1 was liberated from the HCO present in BA medium due to consumption of the bicarbonate buffer. This further leads to a decrease of the H.sub.2:CO.sub.2 ratio, similar to the observations of other authors (see
[0097] Gas fermentation with M. wolfeii BSEL1 is extended to other CO.sub.2 industrial streams such as syngas or combustion flue gases. Interestingly, methanogens could also be used as biocatalyst to transform CO.sub.2 into CH.sub.4 during space missions. For instance, CO.sub.2 could be sequestrated from the Martian atmosphere or from the exhaust breath of astronauts and transformed into CH.sub.4. The resulting CH.sub.4 could later be liquified and used as rocket fuel or as an alternative source for electricity generation in microturbines.
Materials and Methods
Anaerobic Medium Preparation (BA Medium and Wastewater)
[0098] Unless specified differently, BA medium consisted of the following components [mg/L]: NH.sub.4Cl [1,000]; NaCl [100]; NaHCO.sub.3 [2,600], MgCl.sub.2*6H.sub.2O [100]; CaCl.sub.2*2H.sub.2O [50]; K.sub.2HPO.sub.4*3H.sub.2O [400]; H.sub.3BO.sub.3 [0.1]; ZnCl.sub.2 [0.1]; CuCl.sub.2*2H.sub.2O [0.04]; MnCl.sub.2*2H.sub.2O [0.04]; (NH.sub.4).sub.2MoO.sub.4*4H.sub.2O [0.1]; AlCl.sub.3*6H.sub.2O [0.1]; CoCl.sub.2*6H.sub.2O [0.1]; NiCl.sub.2*6H.sub.2O [0.1]; ethylenediaminetetraacetic acid Na-salt (EDTA) [0.5]; Na.sub.2SeO.sub.3*5H.sub.2O [0.1]; FeCl.sub.2*4H.sub.2O [2]; biotin [0.02]; folic acid [0.02]; pyridoxine hydrochloride [0.1]; riboflavin [0.05]; thiamine hydrochloride [0.05]; cyanocobalamin [0.001]; nicotinic acid [0.05]; p-aminobenzoic acid [0.05]; lipoic acid [0.05]; DL-pantothenic acid [0.05]; and resazurin sodium salt [0.5]. BA medium was made anoxic following modified Hungate techniques: Distilled water was used for medium and stock solutions preparation. Distilled water was pre-reduced by boiling for at least 5 min. The medium was prepared in a flask with a constant flow of pure N.sub.2 delivered through a blunt end needle to minimize air infiltration into the flask during medium preparation. The reducing agent, L-cysteine HCl*H.sub.2O was added to a final concentration of 0.53 g/L. The reduced minimal medium was transferred in aliquots of 4.5 mL to Balch tubes previously flushed with N.sub.2. The tubes were sealed with chlorobutyl rubber stoppers and aluminum crimp seals. The tubes were pressurized to 134.31 kPa with pure N.sub.2 and autoclaved at 121 C. for 20 min. Before use, the medium was given Na.sub.2S (0.06 g/L) and vitamins in the form of filter-sterilized stock solutions. YE added for testing was bacteriological grade and was acquired from Avantor, USA.
[0099] The initial inoculant for isolation was taken from a thermophilic anaerobic digester that was operated in our laboratory originally seeded with manure digestate and fed with sewage sludge as raw material with a retention time of 15 days.
[0100] To determine the effect of pH on the specific growth rate of M. wolfeii BSEL1, bicarbonate in BA medium was replaced with a bicarbonate-carbonate buffer (0.04 M). The amounts of carbonate and bicarbonate to prepare this buffer were calculated using Henderson-Hasselbalch equation. The pH was adjusted using various partial pressures of CO.sub.2 provided to the Balch tubes while maintaining a constant partial pressure of H.sub.2 (166.44 kPa) among the different treatments. The respective partial pressures of CO.sub.2 were: 71.4 kPa (pH 7.0), 41.4 kPa (pH 7.4), 26.9 kPa (pH 7.8), 19.3 kPa (pH 8.3), and 14.5 kPa (pH 8.5). To adjust the pH to 6.5, the medium was added a lower concentration of bicarbonate (0.02 M) and no carbonate was added. In every case, the pH of the medium was verified in abiotic controls after incubation at 64 C. for 24 h. pH measurements were performed at near 64 C.; the pH measurement at room temperature was discarded as it is not representative of the pH at culture conditions in this case because the temperature changes interfere with the solubility of CO.sub.2. Thus, the pH readings were compensated from the solution temperature to standard temperature (25 C.) using a pH temperature correction calculator. In preliminary tests, it was determined that a simpler approach for adjusting the pH by addition of NaOH (2 M) and HCl (2 M) during the medium preparation was not effective to maintain the pH above 7.3 after incubation with CO.sub.2, likely due to the formation of carbonic acid, when CO.sub.2 dissolves in water (v+H.sub.2O.fwdarw.H.sub.2CO.sub.3.fwdarw.H.sup.++HCO.sup.), which acidifies the medium. In every case, the tubes were incubated in a horizontal position at 160 rpm (3 cm orbit diameter) to maximize gas-liquid mass transport.
[0101] The wastewater used as alternative for the culture medium was obtained by centrifugation at 4,000 g for 10 min of digested sewage sludge sampled from Walla Walla wastewater treatment facility (Washington State, USA). The supernatant (wastewater) was collected, then boiled for 5 minutes, and added with L-cysteine HCl*H.sub.2O to a final concentration of 0.53 g/L. The pre-reduced wastewater was autoclaved at 121 C. for 30 min in Balch tubes that were previously flushed with N.sub.2. Na.sub.2S was added to a final concentration of 0.06 g/L from a filter-sterilized stock solution.
Pressure Drop and Gas Consumption in Balch Tubes
[0102] The pressure in the Balch tubes was measured using a pressure gauge (Grainger, 4FLV8, USA) modified to allocate a Luer-Lok fitting. With this fitting, the pressure gauge was able to tightly hold a sterile hypodermic needle that was later used to penetrate the rubber stopper on the Balch tubes and measure the pressure within. The tubes were allowed to cool down to room temperature for 30 min prior to every pressure measurement. After every pressure measurement, the enrichment cultures were re-pressurized with the 70/30 H.sub.2:CO.sub.2 mixture (134.31 kPa absolute pressure) and returned to the incubator. In every generation, a blank without inoculation (abiotic control) was included to detect and subtract the contribution of gas solubilization to the pressure drop. The gas consumption (Gi) during the enrichment cultivation was calculated from the pressure drop (A P) in the Balch tube using Eq. 1, where P.sub.Abiotic is the pressure drop due to gas solubilization (obtained from the abiotic control), V.sub.H is the volume of the headspace in the Balch tube, T.sub.S is the standard temperature for gases (273.15 K), T.sub.R is the room temperature (ca. 293.15 K), and P.sub.S is the standard pressure for gases (1 atm). The use of a 70/30 H.sub.2:CO.sub.2 mixture in the batch cultures, instead of a stoichiometric mixture 80/20, was to avoid the formation of pressures lower than atmospheric in the Balch tubes and prevent potential air in-leakage into the tubes.
16S rRNA Gene Sequencing and Alignment
[0103] DNA extraction was performed via crude alkaline lysis with 0.2 M NaOH (1% SDS, 30 seconds of incubation) from a pellet of overnight grown M. wolfeii BSEL1 cells; the DNA extract was directly used in PCR. The 16S rRNA gene amplification was performed using two different sets of PCR primers: 40F (5-GAT TAA GCC ATG CAA GTC GAA CGA-3) (SEQ ID NO: 1), 765R (5-CAT CGT TTA CGG CCA GGA CTA C-3) (SEQ ID NO: 2), 450F (5-CTT CTG GAA TAA GGG CTG GGC A-3) (SEQ ID NO: 3), 1430R (5-CTC CTC AAA GAA CCC AGA TTC GAC-3) (SEQ ID NO: 4). Following amplification, enzymatic cleanup was performed using Exonuclease IShrimp alkaline phosphatase. The amplicons were dye-terminator Sanger sequenced in a 3730xl DNA Analyzer (Applied Biosystems) according to the manufacturer instructions. The primer sequences were trimmed from the raw chromatograms. The cured forward sequences were further validated against their reverse complementary homologs, 100% matches were obtained between forward and reverse sequences. The validated sequences were initially compared with the NCBI library 16S Microbial Sequences using BLAST 2.7.1+ for pre-identification. The evolutionary history of M. wolfeii BSEL1 was inferred using the maximum likelihood method and Tamura-Nei model. This analysis involved the 15 nucleotide sequences of the known species in the Methanothermobacter genus.
[0104] There was a total of 1,507 positions in the final data set. The evolutionary analysis was conducted in MEGA11.
Whole-Genome Mapping
[0105] Genomic DNA was extracted from a frozen stock of M. wolfeii BSEL1 using bead-beating for cell lysis and a silica column for genomic DNA purification. DNA sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (PCR-free) (NEB, USA) for Illumina, following the manufacturer instructions. The genomic DNA was fragmented by acoustic shearing with a Covaris S220 instrument. Fragmented DNA was cleaned up and end repaired. Adapters were ligated after adenylation of the 3 ends followed by enrichment by limited cycle PCR. DNA libraries were validated using a High Sensitivity D1000 ScreenTape on a TapeStation (Agilent, USA) and were quantified using a Qubit 2.0 Fluorometer.
[0106] The sequencing library was clustered onto a flow cell on a MiSeq instrument (Illumina, USA) according to the manufacturer instructions. The sample was sequenced using a 2250 bp paired-end configuration. Image analysis and base calling were conducted by the Illumina Control Software. Raw sequencing data were converted into fastq files using Illumina bcl2fastq 2.20. One mismatch was allowed for index sequence identification.
[0107] FastQC v.0.12.1 was used to verify the quality of the sequencing data before and after quality trimming. Four trimming operations were conducted using Trimmomatic v.0.38 to remove low-quality and short reads. The trimming operations involved leading and trailing (3 bp length), as well as sliding window trimming averaging across four bases and a quality threshold of 15. Reads under 30 bp were also removed. Quality check and data curation were conducted in a Galaxy environment. The trimmed reads were then de novo assembled using SPAdes v.3.10 with a minimum contig length of 1,000 bp and SKESA v.2.4 with hash count enabled (13 contigs were assembled). Annotation was conducted using NCBI PGAP with taxonomy check enabled. Genome assembly and annotation were performed in a WSL Ubuntu 22.04.2 LTS operating system. PGAP dependencies were stored and run in a Docker container. The completeness of M. wolfeii BSEL1 genome was estimated using PGAP against the default model organism for the Methanothermobacter genus. The circular genomic map was built in Proksee.ca.
Growth Assays and Modeling
[0108] The growth curves in the various conditions were obtained by monitoring the optical density (=590 nm) within the Balch tubes using a spectrophotometer TS Genesys 20 (Thermo Scientific, USA) equipped with a tube holder. Blanks with sterile growth medium were used to adjust the spectrophotometer to zero. During the test with YE and vitamins, the spectrophotometer was adjusted to zero using a specific blank for each treatment to avoid the loss of optical definition while using mediums with various compositions. Linear correlation was established between the optical density readings and dry biomass concentration x (R.sup.2=0.9912). x was measured using gravimetry. The logistic population growth model (equation 2) was fitted to the biomass concentration measurements during exponential phase of M. wolfeii BSEL1. The specific growth rate was equal to the slope of the linearized growth curve (equation 3). The duplication time td in the various conditions was calculated using Eq. 4. Prior to every assay, M. wolfeii BSEL1 wild type was cultivated in a serum vial until exponential phase was reached to be transferred into the Balch tubes. The inoculation order during every assay was randomized.
Microscopy
[0109] Phase contrast and fluorescent microscopy were performed on a DMI6000B inverted microscope (Leica, Germany). For fluorescence microscopy, the cells were observed immediately after centrifugation at 4,000 g for 10 min. The microscope was equipped with a filter cube 19001-AT (Chroma, USA) which had a 420/40 excitation filter (T: 42020 nm) and a 465 nm long pass barrier/emission filter (T: 465 nm+), corresponding with the excitation and emission wavelengths of the F420 cofactor, respectively.
Gas Analysis
[0110] Gas composition was measured in a mass spectrometer UGA 100 (SRS, USA) equipped with an ionizer, a quadrupole, and a Faraday cup detector. The peaks at 2, 15, and 44 atomic mass units (amu) were used for H.sub.2, CH.sub.4, and CO.sub.2 quantification, respectively. The signal at 16 amu was not appropriate for CH.sub.4 analysis as it overlaps with the response to an ionic fragment of CO.sub.2 in the mass spectrum. Standard curves were developed using standard gases (Oxarc, USA) diluted in N.sub.2. The calibration gas mixtures for this purpose were calculated using Dalton law. The gases volumetric compositions and the mass spectrometer signals had correlation coefficients of >0.99.
Bioreactor Used for CO.SUB.2 .Fermentation
[0111] A non-stirred 8 L cylindrical bubble column reactor made of stainless steel was used to cultivate M. wolfeii BSEL1 with a constant pressure of 134.31 kPa. The pressure was analogically controlled by a pressure switch (Tameson, UK) connected in feedback loop with a solenoid valve (Parker, USA). The temperature was controlled by a temperature controller (SOLO 4848, AutomationDirect, USA) connected to a heating belt, covering the outer surface of the bioreactor, and a thermocouple installed on the lid of the bioreactor, sensing the temperature at half of the bubble column inside the bioreactor. The recirculation superficial gas velocity (2 cm/min) was powered and controlled using a peristaltic pump (Watson-Marlow 520S, USA). A carbonation stone located at the bottom of the reactor was connected to the gas distribution line. A pressurized storage tank was used to distribute the gas substrate to the reactor. The system relied on Tri-clamp, Swagelok, Luer-lock, and barbed fittings in order to maintain gas tightness. The gas substrate mimicked a biogas:H.sub.2 mixture (26% CH.sub.4, 13% CO.sub.2, 61% H.sub.2) with a H.sub.2:CO.sub.2 ratio equal to the stoichiometric ratio required for hydrogenotrophic methanogenesis (CO.sub.2+4 H.sub.2.fwdarw.CH.sub.4+2 H.sub.2O).
Statistical Analyses
Kruskal-Wallis tests and Conover post hoc tests were conducted in R (4.4.1).
[0112] While the invention has been described in terms of its several exemplary embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.