Field of the invention

The present disclosure relates to a composition, a method and use thereof for reducing atmospheric content in methane. In particular, the invention is suitable for alleviating environmental impact of methane released from permafrost when undergoing prolonged periods of temperature above 0°C.

Background of the invention

Ground is storing about 4 Eg (i.e. 4 x 10 ¹⁸ g) of carbon globally, out of which 40 % are located in permafrost soils in the circumpolar areas, according to the IPCC (International Panel on Climate Change, United Nations Environmental Program, Geneva, Switzerland) from online report https://www.ipcc.ch/site/assets/uploads/2018/02/WG1AR5_all_f inal.pdf, also available on paper: IPCC, 2013: Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change [Stocker, T.F., D. Qin, G.-K. Plattner, M. Tignor, S.K. Allen, J. Boschung, A. Nauels, Y. Xia, V. Bex and P.M. Midgley (eds.)]. Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA, 1535 pp.

Permafrost is defined as soil, rock or sediment that is frozen for more than two consecutive years. A noticeable part of permafrost soil is mostly acidic, located in arctic latitudes, i.e. above approximatively 66° 33’ N coordinate and associated to tundra and taiga ecosystems. Gases trapped in taiga and tundra permafrost include CO ₂ (carbon dioxide) and CH ₄ (methane). These gases are released in the atmosphere when permafrost melts. Other permafrost areas include icy lands such as Greenland and Antarctica and significant mountains and highlands such as Himalaya, Alps, northern part of the Rocky Mountains, Svalbard archipelago, etc.

Considering methane is four times more potent greenhouse gas than carbon dioxide, there is a need to limit release thereof in the atmosphere.

Methane and carbon dioxide in taiga and tundra is principally biogenic and produced by microorganisms such as yeasts, bacteria and fungi. Among those microorganisms, methanotrophs, which are microorganisms using methane as carbon feedstock for growing, are present and are elsewhere studied for biotechnological applications. However, until now, most of the studied microbes required mild to high temperature to grow, complex media (metals, inorganic compounds, vitamins) and methane pressure barely encountered in nature (Methanotrophs, Springer, 2019, and www.methanotroph.org).

The difficulty in using methanotrophs for environmental purpose is that the methane oxidation must occur at low methane concentration (hundreds of ppm) and usually at mild to low temperature (close to 0°C). This cannot be achieved by conventional methanotrophs. Microbes able to grow at low temperature and on low methane pressure are known to exist since several decades (Whalen et al. Nature. 1990 and Oremland et al, Nature, 1992), but it is only very recently that one of them, Methylocapsa gorgona, has been isolated and studied and its genome sequenced (Tveit et al. PNAS. 2019). This bacterium has a “particulate” methane monooxygenase (pMMO) with a higher specific affinity for ChU than any other studied pMMO. Experimental data showed that M. gorgona can use ChU, CO2, O2 and N2. Furthermore, it has the genes allowing the use of CO and H2 as energy source. However, growth of M. gorgona is very slow since months of cultivation are needed to obtain a colony on polycarbonate filter floating on Nitrate Minimal Salts (NMS) medium. In addition, this bacterium showed an optimal temperature of 37°C suggesting that other methanotroph could have a better adaption to cold environment. Some metagenomics studies of permafrost are available since few years (Singleton et al. The ISME journal, 2018), but more experimental data on these microbes and their synergies are needed.

Microbes spreading in the environment for decontamination purposes has been proposed to remove from soil nitrates or various xenobiotic compounds (Methanotrophs, Springer, 2019, Preininger et al, AMB 2018). Methanotrophs have also been used to reduce the methane emission in landfill, crop and animal husbandries (Methanotrophs, Springer, 2019, Davamani et al, Sci Total Environ. 2020 Huang et al, Sci Total Environ. 2019) but very little data are available on using methanotrophs in permafrost.

Using methanotroph to remove CH ₄ emission from permafrost requires microbes fixing low concentration of methane and also able to capture the gases and grow at low temperature. In addition, the surface area susceptible to release methane is immensely vast and might be difficult to access.

Cited references

Davamani V, Parameswari E, Arulmani S. Mitigation of methane gas emissions in flooded paddy soil through the utilization of methanotrophs. Science of the total environment. 2020. Huang D, Yang L, Ko JH, Xu Q.. Comparison of the Methane-Oxidizing Capacity of Landfill Cover Soil Amended With Biochar Produced Using Different Pyrolysis Temperatures. Science of the total environment. 2019. Oremland RS, Culbertson CW. Importance of methane-oxidizing bacteria in the methane budget as revealed by the use of a specific inhibitor. Nature. 1992.

Oremland RS, Capone DG, Use of “Specific” Inhibitors in Biogeochemistry and Microbial Ecology. Chapter in book: Advances in Microbial Ecology. 1988.

Ross MO, MacMillan F, Wang J, Nisthal A, Lawton TJ, Olafson BD, Mayo SL, Rosenzweig AC, Hoffman BM. Particulate Methane Monooxygenase Contains Only Mononuclear Copper Centers. Science. 2019.

Singleton CM, McCalley CK, Woodcraft BJ, Boyd JA, Evans PN, Hodgkins SB, Chanton JP, Frolking S, Crill PM, Saleska SR, Rich VI, Tyson GW. Methanotrophy Across a Natural Permafrost Thaw Environment. The ISME Journal. 2018.

Tveit AT, Hestnes AG, Robinson SL, Schintlmeister A, Dedysh SN, Jehmlich N, von Bergen M, Herbold C, Wagner M, Richter A, Svenning MM. Widespread Soil Bacterium That Oxidizes Atmospheric Methane. PNAS. 2019.

Whalen SC, Reeburgh WS. Consumption of atmospheric methane by tundra soils. Nature. 1990.

Book: Methanotrophs. Microbiology Fundamentals and Biotechnological applications. Editor: Eun Yeol Lee. Springer. 2019.

Summary of the invention

According to a first aspect, the invention relates to a composition for the decomposition of methane comprising

(a). at least one methanotroph microorganism or spore thereof apt to decompose methane when said methanotroph microorganism or spore thereof is cultivated,

(b). a porous solid phase support substantially deprived of toxicity towards said methanotroph microorganism, wherein the porous solid phase support comprises particles having a mean porous surface greater than 20 m ² /g, and true density of at most 4 g/cm ³ , preferably at most 2 g/cm ³ and mean particle size from 20 pm to 15 mm, preferably from 100 pm to 5 mm.

A mean porous surface greater than 20 m ² /g may facilitate the impregnation of the methanotroph microorganisms on the porous solid phase support, as well as the growth of the microorganisms on the porous solid phase support.

Such a low true density allows the flotation of the porous solid phase support when immerged in water, for example in a wetland or upon thawing of a soil, in particular upon thawing of permafrost. This allows the methanotroph microorganisms which require aerobic conditions to oxidize methane to always remain in contact with air whatever the temperature of the soil.

Mean particle size from 20 pm to 15 mm, preferably from 100 pm to 5 mm, may ease the dispersion/spreading of the composition in an environment zone.

The porous solid phase support preferably comprises particles of activated carbon having a mean porous surface greater than 150 m ² /g, preferably greater than 400 m ² /g.

The porous solid phase support comprises particles of at least one of diatomaceous earth, zeolite, clay, volcanic rock, limestone, sandstone, optionally roasted wood chips, sawdust, lichen, moss and biochar, preferably biochar.

The composition according to the invention preferably further comprises

(c). a microorganism growth medium comprising at least 5 ppm of Fe and/or Cu, optionally a N source, at least 5 ppm of P, optionally a binder or carrier vehicle which is a gel or a gum, said gel or gum comprising (i) oligomers and/or polymers of carbohydrates such as carrageenan, agar, agarose, noble agar, alginic acid or alkaline salt thereof, guar, carob, glucomannan, gellan gum, xanthan and/or (ii) oligomers and/or polymers of amino-acids such as gelatin and/or (iii) phytagel ™ or silica gel.

The at least one methanotroph microorganism or spore thereof express, when cultivated, an enzyme apt to oxidize methane, such as a methane monooxygenase. The methane monooxygenase is preferably selected among EC 1.14.18.3 and EC 1.14.13.25, preferably 1.14.18.3.

Each of the second and/or first methanotroph microorganism is advantageously independently selected among methane oxydating bacteria genera Methylocapsa, Methylocella, Methylobacterium, Candidatus Methyloaffinis, Candidatus Methylospira, Candidatus Methylomirabilis, Beijerinckia, Methyloferula, Methylocystis, Methylosinus, Methylococcus, Methyloglobulus, Methylomonas, Methylosarcina , Methylogaea, Methylosoma, Methyloprofundus, Methylovulum, Methylosphaera, Methylocaldum, Methylmarinovum, Methylomarinum, Methylothermus, Methylomicrobium, Methylotuvimicrobium, Methylotyvimicrobium, Methylobacter, Methyloceanibacter, Methylohalobius , preferably Methylocapsa. The methanotroph microorganism is preferably a Methylocapsa species chosen among Methylocapsa gorgona, Methylocapsa acidiphila, Methylocapsa aurea, Methylocapsa palsarum and Methylocapsa silvestris, preferably Methylocapsa gorgona. These methanotroph microorganisms do not require high concentration of methane such that they are able to oxidize methane present in the atmosphere, even at low concentration.

According to a second aspect, the invention relates to the use of the composition according to one any of the embodiments according to its first aspect, for mitigating methane release from the environment into the atmosphere.

The environment is preferably chosen among a permafrost area such as taiga or tundra, a wetland, a wastewater treatment unit, a digestive tract of a mammal.

The invention according to its second aspect may be used as food additive for vertebrate animals, preferably human or ruminant.

The invention according to its second aspect may be used for depolluting air within closed volumes such as in home, office, shop, workshop, factory, cowshed, stable, storage of anaerobic digestion leachate, and in any enclosed space welcoming public such as mall, museum or sport arena.

The invention according to its second aspect may be used as fertilizer or as soil remediation mean.

According to a specific embodiment of the second aspect of the invention, methane release zone is identified using spectroscopic or spectrometric means prior to dispersal of the composition within the said identified methane release zone.

According to a third aspect, the invention is about a method for the mitigation of atmospheric methane release from the soil within an environment zone, comprising

(i) identifying a location within the environment zone susceptible of methane release,

(ii) collecting a soil sample containing at least one methanotrophic microorganism within said zone, either:

(iii) inoculating the soil sample of step (ii) into an appropriate cultivation medium under appropriate cultivation conditions to obtain a culture enriched with the at least one methanotrophic microorganism, optionally in the presence of other microorganisms, (iv) impregnating a porous solid phase support having a mean porous surface greater than 20 m ² /g, and true density of at most 4 g/cm ³ , preferably at most 2 g/cm ³ and mean particle size from 20 pm to 15 mm, preferably from 100 pm to 5 mm with the culture obtained at step (iii), and

(v) spreading the culture impregnated porous solid of step (iv) within the said environment zone, or:

(vi) inoculating the soil sample of step (ii) into an appropriate cultivation medium under appropriate cultivation conditions to obtain a culture enriched with the at least one methanotrophic microorganism, optionally in the presence of other microorganisms, and in the presence of a porous solid phase support having a mean porous surface greater than 20 m ² /g, and true density of at most 4 g/cm ³ , preferably at most 2 g/cm ³ and mean particle size from 20 pm to 15 mm, preferably from 100 pm to 5 mm to obtain an spent cultivation medium and a culture impregnated porous solid,

(vii) optionally collecting the resulting culture impregnated porous solid of step (vi), and (viii) spreading the culture impregnated porous solid of step (vii) or the combination of the spent cultivation medium and of the culture impregnated porous solid of step (vii) within the said environment zone.

Suitable means for spreading the culture impregnated porous solid include any available transportation and spreading apparatuses such as manned or unmanned flying vessels like helicopters, airplanes or drones, industrial or agricultural vehicles like tractors equipped with tanks, pumps and spreading devices, or hand spreading devices.

Culture impregnated porous solid may be (i) either spread as is on the area to be treated using continuous air jet or air pulse or (ii) suspended in a liquid e.g. water, pumped and spread through an appropriate nozzle.

For optimal dispersion it is desirable that the mean particle size distribution be as narrow as possible and in the range 10-500 pm. Particles must be small enough to allow fair spreading onto the surfaces to be treated and big enough to avoid excessive dispersion in case of e.g. wind gusts. Similarly, particles true density should be adapted to the nature of the soil wherein treatment is desired. Ideally, particles true density should be close to the true soil density so that methanotroph microorganisms can have access to a sufficient amount of air. Particle true density must not be too low to avoid excessive particles washout and/or lixiviation of methanotroph microorganisms in case of runoff water.

In average, a +/- 0,1 difference between particle true density and soil true density is acceptable.

In this third aspect of the invention, the culture impregnated porous solid of step (iv), the culture impregnated porous solid of step (vii) or the combination of the spent cultivation medium and of the culture impregnated porous solid of step (vi) each form a composition for the decomposition of methane as defined with respect of the first aspect in the present invention.

In the invention according to its third aspect, the environment zone may advantageously be a permafrost area such as taiga or tundra or a wetland, preferably a permafrost area.

In the invention according to its third aspect, the porous solid phase support in step (iv) or (vi) may be defined as in the composition of the invention.

In the invention according to its third aspect, the methanotroph microorganism or spore thereof and/or the enzyme apt to oxidize the methane expressed by said methanotroph microorganism or spore thereof, may be as previously defined with respect of the composition herein described.

In the invention according to its third aspect, the cultivation conditions in steps (iii) or (iv) may include: a microorganism growth medium comprising at least 5 ppm of Fe and/or Cu, optionally a N source, at least 5 ppm of P, optionally a binder or carrier vehicle which is a gel or a gum, said gel or gum comprising (i) oligomers and/or polymers of carbohydrates such as carrageenan, agar, agarose, noble agar, alginic acid or alkaline salt thereof, guar, carob, glucomannan, gellan gum, xanthan and/or (ii) oligomers and/or polymers of amino-acids such as gelatin and/or (iii) phytagel ™ or silica gel.

Definitions

Environment: all the elements including physical parameters (temperature, hygrometry if applicable, irradiance, pressure, etc.) that are surrounding a subject of study. It includes organic chemical containing material (living or dead organisms, chemicals and pollutants, etc.) and inorganic material (minerals, inorganic chemicals, water, air components, etc.), whether stable or radioactive.

Methanotroph: means “which likes methane”. In the present document, it relates to a microorganism which is capable of methane oxidation.

Methanogen: means “which produces methane”. In the present document, it relates to a microorganism which is capable of methane generation. Biochar is a porous carbonaceous material obtained by the pyrolysis of biomass, preferably waste vegetal, preferably at a temperature from 350 to 700°C, containing from x to y % of C and z to t % of H along with other elements.

Na2EDTA stands for the disodium salt of Ethylene Diamine Tetra Acetic acid.

A porous solid phase support is substantially deprived of toxicity towards said methanotroph microorganism when no more than half of the mass of the methanotroph microorganism is decomposed by the porous solid phase support for a period of one month in the conditions of the environment in which the methanotroph microorganism is used. The decomposition can be determined by measuring the quantity of methane decomposed during this period.

The mean porous surface (i.e. surface area) may be measured by intrusion mercury porosimetry. The mean porous surface thus corresponds to the external surface and internal surface (total pore area accessible from the surface) of a material. This measurement method is widely used in petroleum refining industry for quantifying solid phase supported catalysts porosity and can be used herein without specific adaptation.

The true density is defined as the quotient of mass over the volume of a sample, without considering pores (accessible or inaccessible) and interparticle space in the material (true volume). In other words, the true density is the density of the material that constitute the particle, without porosity. The true density may be measured by helium pycnometry.

The true density is to be distinguished from bulk density or skeletal density. Bulk density corresponds to the quotient of mass over the bulk volume of the sample. Bulk volume, which is dependent on the particle packing, is the macroscopic volume of a sample that includes all pore spaces and interparticle spaces. Skeletal density corresponds to the quotient of mass over the skeletal volume of the sample. The skeletal volume excludes all spaces that are accessible to the outside but includes the volume of all closed pores that are physically inaccessible. In contrast, true volume only consists of the actual volume of the molecules or atoms that make up the material and excludes all pores (accessible and inaccessible) and interparticle spaces.

The mean particle size may be measured by laser diffraction.

Detailed description of the invention

Examples The embodiments of the present invention will be better understood by looking at the different examples, below.

Example 1: Batch production of a pool of microorganisms isolated from marchland, out of which some are oxidizing methane.

A batch culture containing methanotroph microorganism can be obtained according to the following procedure:

1L of a first sterile liquid mineral media comprising 0.25-1 g/L KNO ₃ , 0.1-1 g/L MgS0 ₄ .7H ₂ 0, 0.717 g/L Na ₂ HP0 ₄ .12H ₂ 0, 0.272 g/L K ₂ HP0 ₄ , 0.1-0.2 g/L CaCI ₂ .6H ₂ 0, 0.005 g/L FeSC> ₄ .7H ₂ 0 dissolved in sterile water is introduced in a 5L sterile bioreactor equipped with stirring under an atmosphere consisting of a sterile air mixture having 5-50%vol. of ChU and 1-5%vol. of C0 ₂ at atmospheric pressure.

1mL of a second sterile liquid mineral media comprising 0.5 g/L Na ₂ EDTA, 0.03 g/L H ₃ BO ₃ , 0.01 g/L ZnS0 ₄ .7H ₂ 0, 0.03 g/L MnCI ₂ .4H ₂ 0, 0.02 g/L CoCI ₂ .6H ₂ 0, 0.03 g/L CuS0 ₄ .5H ₂ 0, 0.002 g/L NiCI ₂ .6H ₂ 0, 0.003 g/L Na ₂ Mo0 ₄ .2H ₂ 0 dissolved in sterile water, is further added into the sterile bioreactor.

10 g of a methanotroph bacteria containing soil sample collected on a marshland is dispersed within the first and second sterile liquid mineral media under stirring.

The bioreactor can be run from 5 to 37°C, preferably in the absence of natural or artificial light and culture growth may be monitored by measuring UV absorbance of aliquots, or by measuring methane consumption using e.g. gas chromatography or infrared spectroscopy.

Traditionally, depending on culture conditions and microorganism species and their combinations, exhaustion of nutrients from the culture medium and methane consumption may vary from 1 week to 3 months.

Gas atmosphere within the bioreactor may be replaced with the fresh sterile air mixture depending on growth rate, usually once after the first month of cultivation, then every week. Alternatively, continuous introduction of fresh sterile air mixture may be used along with degassing.

Ideally, high contact surface between gas and liquid may be achieved using strong stirring and/or bubbling of the sterile air mixture using a circulating pump, wherein a pump exhaust is preferably positioned at the bottom of the reactor and wherein the gas that leaves the exhaust passes through an outlet device that is designed to release tiny bubbles into the liquid phase via the use of an array of small apertures.

Culture may be isolated through centrifugation or filtration on filters or membranes having porosity beyond average size of cultivated microorganisms. Typically, filters or membranes suitable for bacteria retention have 0.22-0.45 pm pore size.

Alternatively, culture may be used without further processing, depending on the application.

Example 2: Batch production of a solid phase supported pool of microorganisms isolated from marchland, out of which some are oxidizing methane.

Procedure of example 1 is reproduced except that 100 g of commercial biochar obtained by pyrolysis of vegetal waste can be introduced into the bioreactor at any time before and/or during microorganism cultivation, preferably before microorganism cultivation.

Example 3: Isolation of methanotrophic microorganism from a sample isolated from tundra permafrost.

Procedure from example 1 may be reproduced except that a sample of permafrost tundra soil is used in lieu of marchland sample. No biochar is added in the bioreactor. An aliquot is sampled after at least 1 week. This aliquot is diluted in physiological serum and an aliquot of the resulting diluted solution is streaked onto sterile agar based growing medium contained in petri dishes, according to routine microbiological procedures. Cultivation is carried out at different temperature (5, 15, 25 and 35°C) under methane atmosphere as per example 1 until at least one single microorganism colonies become apparent. Those single microorganism colonies may be each identified and characterized using conventional PCR techniques known to the skilled artisan.

Example 4: Batch production of a solid phase supported single microorganism species isolated from tundra permafrost soil sample, which is oxidizing methane.

Single colonies of interest from example 3 may be separately cultivated in a bioreactor using the full procedure of example 1 or 2 to provide optionally biochar supported, single microorganism species.

Example 5: Solid phase supported culture of methanotroph microorganism 10 g of a sample of soil containing a pool of methanotroph microorganisms isolated from a landfill may be suspended in 50 ml_ of the first and second sterile liquid media mixture of example 1, and shaken vigorously using a mechanical disperser such as those sold by IKA under Ultra-Turrax® brand name. Resulting suspension is filtrated to remove particulates that might clog spraying device and 1 ml_ of subsequent filtrate is sprayed over 100 cm ² of commercial polycarbonate filter in a 1 L sterile closed bottle. 0,1 L of the first and second liquid media mixture of example 1 is slowly introduced into the bottle so that the polycarbonate filter is kept floating on the liquid. Gas atmosphere inside the bottle, temperature and cultivation duration may be set according to example 1 or adapted to the kind of microorganism to be grown. For instance, if one wishes to favor growth of microorganisms such as Methylococcus capsulatus, a gas atmosphere that is deprived or at least sufficiently depleted in oxygen should be used. Cultivation conditions and duration shall be adapted to growth rate of microorganism species to be favored and growth may be monitored according to known techniques, such as those described in PNAS 116, n°17, pp.815-8524 (2019).

Example 6: one pot preparation of ready-to-use porous solid phase supported methanotroph microorganism

10 g of a sample of soil containing a pool of methanotroph microorganisms isolated from a landfill may be suspended in 50 ml_ of the first and second sterile liquid media mixture of example 1, and shaken vigorously using a mechanical disperser such as those sold by IKA under Ultra-Turrax® brand name. The resulting suspension is mixed with 100g of commercial biochar dispersed in a 5L sterile closed vessel to obtain a methanotroph impregnated biochar. The gas mixture composition in the vessel may be similar to the one of example 1. A small amount of the first and second liquid media mixture of example 1 is slowly introduced into the bottle so that the methanotroph impregnated biochar is moistened enough to allow for microorganism growth. On the contrary, care should be taken to avoid excessive moistening to allow easy access of microorganisms to gas atmosphere inside the vessel and maximize contact between gas and solid phase while providing sufficient moistening. In this respect, small amounts of sterile water or of first and second sterile liquid media mixture may be added if it appears the biochar dries. Monitoring of microorganism growth may be achieved using monitoring of methane consumption using any available method. An acceptable available method could be infrared measurement of residual methane present in a sample of the gas phase inside the 5 L vessel or via measurement of combustion enthalpy of gas. Microorganism cultivation may be stopped once methane consumption rate reaches a target freely defined by the operator. In this case, fresh gas mixture addition as well as water and/or first and second sterile liquid media mixture addition are stopped, and a stream of dry nitrogen or dry air is slowly passed inside the vessel until the biochar is a powder again. The biochar is then collected and may be used as such for dispersion over a plot wherein a methane leakage occurs. Alternatively, this biochar may be packed into a cartridge for gas filtration. In this case the cartridge may be useful for the removal of methane contained in the atmosphere of closed spaces before release in the environment through e.g. forced circulation of methane containing gas inside the cartridge.