Microbial compositions and methods for improving biomethane production

Abstract

Compositions and methods for improving biogas production during the treatment of organic waste, such as livestock, municipal, or industrial waste, are disclosed. The invention provides microbial compositions comprising specific genera of bacteria and archaea for enhancing methane production. The compositions are configured for introduction into a bioreactor to enhance the anaerobic digestion process, specifically increasing methane yield and / or accelerating waste degradation.

Description

[0001] MICROBIAL COMPOSITIONS AND METHODS FOR IMPROVING BIOMETHANE PRODUCTION FIELD OF THE INVENTION

[0002] The present invention relates to compositions of microbes and methods for improving biogas production during treatment of waste, such as livestock waste, municipal waste, and industrial waste. Particularly, the invention relates to compositions comprising specific genera of bacteria and archaea for enhancing methane production in a bioreactor during waste treatment and related methods.

[0003] BACKGROUND OF THE INVENTION

[0004] Waste discharge from cities and intensive livestock farms constitutes the main organic pollutant loads into rivers. This wastewater has the potential to cause high levels of environmental pollution and is a significant challenge for Wastewater Treatment Plants (WWTP), into which the waste is often discharged for treatment. The wastewater is characterized by high loads of organic matter, suspended solids, nitrogen, and phosphorus that significantly increase the growth of algae (i.e., algal blooms), which can be harmful because they produce elevated toxins and bacterial growth that cause illness for those who come into contact with the polluted water, consume tainted fish or shellfish, or drink contaminated water.

[0005] Anaerobic digestion (AD) is a biological process that breaks down organic materials (feedstocks) in the absence of oxygen (anaerobic conditions) into biogas, containing about 50% methane (CH4) and 50% carbon dioxide (CO2). The production of biogas and other products of biological processes (collectively referred to herein as "bioproducts" for the sake of brevity), as well as the quality of water effluent and discharged sludge, depends largely on the microbial community in the reactors. Accordingly, the microbial community in the reactors needs to be optimized to increase the yield of biogas and other bioproducts.

[0006] By its ability to replace conventional fuels, biomethane has diverse applications across various sectors. For example, it can be used as a clean and sustainable alternative to natural gas for electricity generation and heating, reducing reliance on fossil fuels and lowering greenhouse gas emissions. In the transportation sector, biomethane can fuel vehicles, offering a greeneroption compared to gasoline or diesel and contributing to reduced air pollution. Moreover, the by-products of the biomethane production process via anaerobic digestion (i.e., digestate) can be utilized as nutrient-rich fertilizers in agriculture. Overall, its versatility in reducing waste, generating energy, and supporting sustainable practices makes biomethane a valuable component of a circular economy. Thus, improving biomethane production is of great importance for advancing environmental sustainability, energy security, and economic development.

[0007] It is therefore an object of the present invention to provide microbial compositions for the improved performance of an apparatus in terms of increased yield of biomethane by anaerobic digestion of waste.

[0008] It is another object of the invention to provide a method for increasing biomethane yield by applying said microbial compositions.

[0009] The above and other purposes and advantages of the invention will become apparent as the description proceeds.

[0010] SUMMARY OF THE INVENTION

[0011] In one aspect of the invention, there is provided a composition for enhancing biomethane production by anaerobic digestion of waste, wherein the composition comprises at least one microbe of each of the genera: Romboutsia, Paeniclostridium, Methanobacterium, Methanothrix, Clostridium sensu stricto 1, midas_g_12, Candidatus Cloacimonas (Ca. Cloacimonas), and Turicibacter.

[0012] According to one embodiment of the invention:

[0013] a) the at least one microbe of the genus Romboutsia comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQID NOs: 1-221; and / or

[0014] b) the at least one microbe of the genus Paeniclostridium comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 222-339; and / or

[0015] c) the at least one microbe of the genus Methanobacterium comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 340-478; and / or

[0016] d) the at least one microbe of the genus Methanothrix comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 479-919; and / or

[0017] e) the at least one microbe of the genus Clostridium sensu stricto 1 comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 920-1319; and / or

[0018] f) the at least one microbe of the genus midas_g_12 comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1320-1455; and / or

[0019] g) the at least one microbe of the genus Ca. Cloacimonas comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1456-1755; and / or

[0020] h) the at least one microbe of the genus Turicibacter comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1756-2024.

[0021] According to another embodiment of the invention, the microbes of each genus are present in the composition at the following ratios:

[0022]

[0023]

[0024] According to a further embodiment of the invention, the composition comprises one or more of the following species: midas_s_34, midas_s_5918, Romboutsia unclassified, midas_s_31016, midas_s_43100, midas_s_59815, Romboutsia lituseburensis, Paeniclostridium unclassified, Paeniclostridium sordellii, Methanobacterium unclassified, midas_s_3006, Methanobacterium formicicum, Methanobacterium lacus, midas_s_9526, Methanothrix_soehngenii, Methanothrix_harundinacea, midas_s_36, Methanothrix unclassified, Methanothrix thermoacetophila, midas_s_94826, midas_s_90667, midas_s_64, Clostridium sensu stricto 1 unclassified, midas_s_3795, Clostridium disporicum, Clostridium butyricum, midas_s_48014, midas_s_101, midas_s_54021, Clostridium chartatabidum, midas_s_57157, Clostridium saudiense, midas_s_8895, Clostridium tertium, midas_s_93503, midas_s_4202, midas_g_12 unclassified, midas_s_3429, midas_s_15546, midas_s_91465, midas_s_301, midas_s_246, Ca. Cloacimonas unclassified, midas_s_3011, midas_s_110004, Turicibacter sanguinis, and Turicibacter unclassified.

[0025] According to a specific embodiment of the invention, said species are present in the composition at the following ratios:

[0026]

[0027]

[0028]

[0029] The composition according to any one of claims 1-5, wherein the composition comprises one or more additional microbe of the genus selected from midas_g_467, Ca. Brevefilum, midas_g_30901, Anaerolinea, midas_g_156, Flexilinea, Leptolinea, p-1088-a5 gut group, Ca. Anammoximicrobium, Christensenellaceae R-7 group, Truepera, Syntrophomonas, midas_g_7812, Lachnospiraceae NK3A20 group, Nocardioides, Corynebacterium, DMER64, Mesotoga, Limnobacter, and midas_g_1946.

[0030] According to a specific embodiment of the invention, the one or more additional microbe is present in the composition at the following ratios:

[0031]

[0032]

[0033] According to one embodiment of the invention, the composition comprises at least a total of lxio9microbial cells per gVS of the composition. In a specific embodiment of the invention, the composition comprises a total of lxio9to lxio12microbial cells per gVS of the composition.

[0034] According to another embodiment of the invention, the composition comprises a total concentration of microbial cells of from lxio7to 2.4xl012cells per 1 g or per 1 ml of the composition.

[0035] In some embodiments of the invention, the composition further comprises additives selected from minerals, growth factors, nutrients, buffers, preservatives, and any combination thereof that contribute to the growth of the microbes.

[0036] In another aspect of the invention, there is provided a method for enhancing biomethane production by anaerobic digestion of waste, the method comprising:

[0037] a) providing waste into a reactor configured to perform anaerobic digestion;

[0038] b) adding a composition according to any one of claims 1-9 to the reactor, thereby obtaining inoculated waste;c) anaerobically incubating the inoculated waste from step (b), thereby generating biogas containing methane; and

[0039] d) collecting said biogas generated in step (c).

[0040] According to one embodiment of the invention, the waste is in the form of sludge.

[0041] In some embodiments of the invention, the composition is added to the reactor once.

[0042] In other embodiments of the invention, the composition is added to the reactor at intervals ranging between once a day and once a month.

[0043] According to a specific embodiment of the invention, the composition is added to the reactor once every two weeks.

[0044] According to some embodiments, prior to adding the composition to the reactor, the composition is dissolved in a biological solution.

[0045] According to one embodiment of the invention, the composition is added to the reactor at a composition-to-waste ratio of from 0.01% to 25% (gVS:gVS).

[0046] According to another embodiment of the invention, the composition is applied to the reactor containing the waste at a volume of from 1 ml to 2.5 L per 10 L of waste or per 10 kg of waste.

[0047] According to yet another embodiment of the invention, the method further comprises: e) removing gases other than methane from the biogas collected in step d).

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Fig. 1 is a schematic representation of an Integrated Ecosystem Solution (IES) system by SGTECH;

[0050] Fig. 2 is a schematic representation of the setup of the biomethane potential laboratory system (BMP test) for assessing the effects of the composition of the invention on biomethane production as employed in Examples 1-3;Fig. 3A shows a schematic representation of a conventional anaerobic digestion system that degrades organic matter (such as livestock manure) into digestate and biogas;

[0051] Fig. 3B shows a schematic representation of the wastewater treatment plant - a facility designed to treat and purify organic matter (wastewater) through physical, chemical, and biological processes, including anaerobic digestion with energy recovery through biogas production;

[0052] Fig. 4 shows the effect of the composition according to one embodiment of the invention, in the form of raw sludge, on biomethane production of municipal wastewater anaerobic sludge of Example 1;

[0053] Fig. 5 shows the effect of a composition according to one embodiment of the invention, in the form of sludge capsule, on biomethane production of municipal wastewater anaerobic sludge of Example 2;

[0054] Fig. 6 shows the effect of a composition according to one embodiment of the invention, in the form of sludge capsule, on biomethane production of livestock waste anaerobic sludge of Example 3;

[0055] Fig. 7 is a schematic representation of the setup for assessing the performance of a target apparatus in a conventional anaerobic continuous stirred-tank reactor (CSTR) system employed in Example 4; and

[0056] Fig. 8 shows the effect of a composition according to one embodiment of the invention, in the form of a sludge capsule, on biomethane production of livestock waste anaerobic sludge in a small-scale CSTR system of Example 4; the arrows indicate administration of the composition to the waste.

[0057] In the Figs.: "FM" is a gas flowmeter; "G" is a generator; "L" is a liter unit; and "U" is a gas upgrader.

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] The microbial compositions according to the invention address the need for improving biomethane production via anaerobic digestion of waste.

[0060] In one aspect of the invention, there is provided a composition for enhancing biomethane production by anaerobic digestion of waste, wherein the composition comprises at least one microbe of each of the genera: Romboutsia, Paeniclostridium, Methanobacterium,Methanothrix, Clostridium sensu stricto 1, midas_g_12, Candidatus Cloacimonas (Ca. Cloacimonas), and Turicibacter.

[0061] As would be appreciated by a person of skills in the art, the term "microbe" refers to a single cell of microbes (i.e., bacteria or archaea). However, for the sake of brevity, the term "microbe" is also used herein to represent one or more populations of microbes of one or more species under the specified genus.

[0062] Accordingly, the compositions of the invention comprise a combination of one or more species of bacteria and / or archaea belonging to each of the 8 genera listed above.

[0063] According to some embodiments of the invention, the microbes in the composition belonging to the genus Romboutsia include, but are not limited to, microbes having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the V4 region of the 16S rRNA encoding gene set forth in SEQ ID NOs: 1-221, each in its own embodiment.

[0064] According to some embodiments of the invention, the microbes in the composition belonging to the genus Paeniclostridium include, but are not limited to, microbes having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the V4 region of the 16S rRNA encoding gene set forth in SEQ ID NOs: 222-339, each in its own embodiment.

[0065] According to some embodiments of the invention, the microbes in the composition belonging to the genus Methanobacterium include, but are not limited to, microbes having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the V4 region of the 16S rRNA encoding gene set forth in SEQ ID NOs: 340-478, each in its own embodiment.

[0066] According to some embodiments of the invention, the microbes in the composition belonging to the genus Methanothrix include, but are not limited to, microbes having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identity to the V4 region of the 16S rRNA encoding gene set forth in SEQ ID NOs: 479-919, each in its own embodiment.

[0067] According to some embodiments of the invention, the microbes in the composition belonging to the genus Clostridium sensu stricto 1 include, but are not limited to, microbes having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the V4 region of the 16S rRNA encoding gene set forth in SEQ ID NOs: 920-1319, each in its own embodiment.

[0068] According to some embodiments of the invention, the microbes in the composition belonging to the genus midas_g_12 include, but are not limited to, microbes having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the V4 region of the 16S rRNA encoding gene set forth in SEQ ID NOs: 1320-1455, each in its own embodiment.

[0069] According to some embodiments of the invention, the microbes in the composition belonging to the genus Candidatus Cloacimonas (Ca. Cloacimonas) include, but are not limited to, microbes having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the V4 region of the 16S rRNA encoding gene set forth in SEQ ID NOs: 1456-1755, each in its own embodiment.

[0070] According to some embodiments of the invention, the microbes in the composition belonging to the genus Turicibacter include, but are not limited to, microbes having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the V4 region of the 16S rRNA encoding gene set forth in SEQ ID NOs: 1756-2024, each in its own embodiment.

[0071] The sequences of the V4 region of the 16S rRNA encoding genes set forth in SEQ ID NOs. 1-2024 were identified using the universal 515F / 806R primers, which are a standardized primer pair widely used for 16S rRNA gene amplicon sequencing (specifically the V4 region) of bacterial and archaeal communities. The sequences were classified according to the MiDAS (Microbial Database for Activated Sludge) 5 database and verified using the mothur softwareprogram (which is a widely used tool for analyzing 16S rRNA gene sequence data) as uniquely characterizing of their relevant genera and species as indicated in the Sequence Listing.

[0072] The term "comprise" and its variations are used herein in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only". Unless the context requires otherwise, this term and its variations are understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group thereof.

[0073] The terms "enhance" and "improve" as used interchangeably herein, including their variations, refer to any increase in the yield of biomethane resulting from anaerobic digestion in the presence of the compositions of the invention compared to the yield of biomethane in anaerobic digestion in the absence of the compositions of the invention.

[0074] The term "waste" as used herein refers to organic matter originating from livestock waste, municipal waste, and industrial waste. Examples of such organic matter include, but are not limited to, animal manure, wastewater biosolids (e.g., municipal sewage sludge), food wastes, residual biomass, crop residues, volatile fatty acids, winery, waste, brewery waste, refinery wastewater, petrochemical industry wastewater, sugar industry waste, slaughterhouse waste, paper and pulp industry waste, refinery effluent Treatment Plant biosludge, or any combination thereof. In the context of the present disclosure, the term "waste" is used interchangeably with the term "sludge".

[0075] As used herein, "16S rRNA" refers to the 1500 bp ribonucleic acid component of the 30S subunit of a bacterial ribosome. The "V4 region" refers to the fourth hypervariable region of the 16S rRNA gene. The " 16S rRNA encoding gene" refers to the genomic DNA sequence that is transcribed to the 16S rRNA product.

[0076] The term "percent identity" or "sequence identity" in the context of two or more nucleic acid sequences refers to the percentage of nucleotides that are identical between a target sequence and a reference sequence (e.g., SEQ ID NOs: 1-2024) when compared and aligned for maximum correspondence over a comparison window. Optimal alignment for comparisonmay be conducted using the local homology algorithm of Smith & Waterman, the homology alignment algorithm of Needleman & Wunsch, or by computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA, or BLASTN). In a specific embodiment of the invention, the alignment is calculated over the full length of the reference sequence defined in the Sequence Listing. It is recognized that a single microbial genome may comprise multiple 16S rRNA operons, which may vary by one or more nucleotides. The presence of at least one operon matching the claimed identity threshold is sufficient to bring said microbe within the scope of the invention.

[0077] Examples of species belonging to the genus Romboutsia include, but are not limited to, midas_s_34, midas_s_5918, Romboutsia unclassified, midas_s_31016, midas_s_43100, midas_s_59815, midas_s_62216, Romboutsia lituseburensis, Romboutsia faecis, Romboutsia hominis, Romboutsia ilealis, Romboutsia sedimentorum, Romboutsia timonensis, midas_s_4289, midas_s_60819, midas_s_72826, midas_s_101269, midas_s_69756, and midas_s_75555.

[0078] Examples of species belonging to the genus Paeniclostridium include, but are not limited to, Paeniclostridium unclassified, Paeniclostridium sordellii, Paeniclostridium ghonii, midas_s_37284, midas_s_46074, midas_s_53879, and midas_s_3225.

[0079] Examples of species belonging to the genus Methanobacterium include, but are not limited to, Methanobacterium unclassified, midas_s_3006, Methanobacterium formicicum, Methanobacterium lacus, midas_s_9526, Methanobacterium aarhusense, Methanobacterium aggregans, Methanobacterium alcaliphilum, Methanobacterium alkalithermotolerans, Methanobacterium arbophilicum, Methanobacterium arcticum, Methanobacterium beijingense, Methanobacterium bryantii, Methanobacterium congolense, Methanobacterium defluvii, Methanobacterium espanolae, Methanobacterium ferruginis, Methanobacterium flexile, Methanobacterium ivanovii, Methanobacterium kanagiense, Methanobacterium mobile corrig., Methanobacterium movens, Methanobacterium movilense, Methanobacterium paludis, Methanobacterium palustre, Methanobacterium petrolearium, Methanobacterium ruminantium, Methanobacterium spitsbergense, Methanobacterium subterraneum, Methanobacterium thermaggregans corrig., Methanobacterium thermalcaliphilum corrig.,Methanobacterium thermautotrophicum corrig., Methanobacterium thermoflexum, Methanobacterium thermoformicicum, Methanobacterium thermophilum, Methanobacterium uliginosum, Methanobacterium veterum, and Methanobacterium wolfei.

[0080] Examples of species belonging to the genus Methanothrix include, but are not limited to, Methanothrix soehngenii, Methanothrix harundinacea, midas_s_36, Methanothrix unclassified, Methanothrix thermoacetophila, midas_s_94826, midas_s_90667, Methanothrix concilii, Candidatus Methanothrix paradoxa corrig., Methanothrix thermophile, midas_s_2347, midas_s_90212, midas_s_90868, midas_s_91051, midas_s_96620, midas_s_98390, midas_s_100784, midas_s_100787, midas_s_100823, midas_s_110840, midas_s_110852, and midas_s_118955.

[0081] Examples of species belonging to the genus Clostridium sensu stricto 1 include, but are not limited to, midas_s_64, Clostridium sensu stricto 1 unclassified, midas_s_3795, Clostridium disporicum, Clostridium butyricum, midas_s_48014, midas_s_101, midas_s_54021, Clostridium chartatabidum, midas_s_57157, Clostridium saudiense, midas_s_8895, Clostridium tertium, midas_s_93503, , Clostridium perfringens, Clostridium paraputrificum, Clostridium chromiireducens, Clostridium saccharoperbutylacetonicum, Clostridium beijerinckii, Clostridium aurantibutyricum, Clostridium sp., Clostridium saccharobutylicum, Clostridium baratii, midas_s_1569, midas_s_36365, midas_s_37035, midas_s_39966, midas_s_41641, midas_s_48765, midas_s_56699, midas_s_57494, midas_s_62978, midas_s_67467, midas_s_78923, midas_s_80153, midas_s_80332, midas_s_86635, midas_s_90787, midas_s_95799, midas_s_99129, midas_s_109078, midas_s_109681, midas_s_109978, midas_s_110147, midas_s_110656, midas_s_112319, midas_s_113170, midas_s_113589, midas_s_113683, midas_s_113986, midas_s_119482, midas_s_119503, midas_s_119836, midas_s_119893, midas_s_3972, and midas_s_59404.

[0082] Examples of species belonging to the genus midas_g_12 include, but are not limited to, midas_s_4202, midas_g_12 unclassified, midas_s_3429, midas_s_15546, midas_s_91465, midas_s_97935, midas_s_113361, midas_s_119386, midas_s_2548, and midas_s_12.Examples of species belonging to the genus Ca. Cloacimonas include, but are not limited to, midas_s_301, midas_s_246, Ca. Cloacimonas unclassified, midas_s_3011, midas_s_110004, Candidatus Cloacamonas acidaminovorans, midas_s_2009, midas_s_7660, midas_s_81840, midas_s_90171, midas_s_90268, midas_s_90627, midas_s_91979, midas_s_93687, midas_s_95016, midas_s_97842, midas_s_104439, midas_s_106877, midas_s_109675, midas_s_110679, midas_s_112235, midas_s_112293, midas_s_112294, midas_s_112889, midas_s_112914, midas_s_113076, midas_s_113465, midas_s_115244, midas_s_115337, midas_s_115467, midas_s_115756, midas_s_116046, midas_s_116092, midas_s_119138, midas_s_119204, midas_s_119290, midas_s_119560, midas_s_120016, midas_s_87, midas_s_1905, and midas_s_2209.

[0083] Examples of species belonging to the genus Turicibacter include, but are not limited to, Turicibacter sanguinis, Turicibacter unclassified, Turicibacter bilis, Turicibacter faecis, and midas_s_43201.

[0084] According to some embodiments of the invention, the microbes in the composition include, but are not limited to, microbes having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the V4 region of the 16S rRNA encoding gene set forth in any one of SEQ ID NOs: 2-221, 223-339, 341-478, 480- 919, 921-1319, 1321-1455, 1457-1755, and 1757-2024, each in its own embodiment, according to Table 1.

[0085] Table 1. Sequences of the V4 region of the 16S rRNA gene.

[0086]

[0087]

[0088]

[0089] In some embodiments of the invention, the combination of microbes in the composition is in the form of individual populations. In other embodiments of the invention, the combination of microbes in the composition is in the form of one or more consortia.

[0090] In one embodiment of the invention, the composition comprises isolates of each species of microbes, which are commercially available or isolated from any source. In another embodiment of the invention, the composition comprises sludge containing the microbes. In a specific embodiment of the invention, the sludge is in the form of a capsule, for example, sludge that was filtered to remove residues of straw, stones, mud, and dirt, and centrifuged to sediment the microbes in the sludge, wherein the capsule is the pellet obtained after the centrifugation step. Optionally, the pellet is then frozen (e.g., at -20°C). According to another specific embodiment of the invention, the sludge is in the form of raw sludge. In another embodiment of the invention, the composition is in the form of a lyophilized powder or liquid culture.

[0091] In one embodiment of the invention, the microbe of each of the above-mentioned genera is present in the composition at a range of from 0.3% to 93% of the entire population of microbes.In a specific embodiment of the invention, the ratio of abundance between the microbes in the composition is as defined in Table 2. It is understood that each numeric value disclosed in Table 1 includes a variation of ±5% of the stated value.

[0092] Table 2. Ratio of abundance between microbes.

[0093]

[0094] According to Table 2, the ratio between the microbes in the composition would be between 21:14:3:29:19:19:17:17 and 43:30:139:125:49:34:176:42 (Romboutsia:Paeniclostridium: Methanobacterium:Methanothrix:Clostridium sensu stricto l:midas_g_12:Ca. Cloacimonas: Turicibacter), including a variation of ±5% of the stated value.

[0095] In a specific embodiment of the invention, the composition comprises one or more species of the genus Romboutsia selected from midas_s_34, midas_s_5918, Romboutsia unclassified, midas_s_31016, midas_s_43100, midas_s_59815, and Romboutsia lituseburensis.

[0096] In another specific embodiment of the invention, the composition comprises one or more species of the genus Paeniclostridium selected from Paeniclostridium unclassified and Paeniclostridium sordellii.

[0097] In yet another specific embodiment of the invention, the composition comprises one or more species of the genus Methanobacterium selected from Methanobacterium unclassified, midas_s_3006, Methanobacterium formicicum, Methanobacterium lacus, and midas_s_9526.In a further specific embodiment of the invention, the composition comprises one or more species of the genus Methanothrix selected from Methanothrix_soehngenii, Methanothrix_harundinacea, midas_s_36, Methanothrix unclassified, Methanothrix thermoacetophila, midas_s_94826, and midas_s_90667.

[0098] In another embodiment of the invention, the composition comprises one or more species of the genus Clostridium sensu stricto 1 selected from midas_s_64, Clostridium sensu stricto 1 unclassified, midas_s_3795, Clostridium disporicum, Clostridium butyricum, midas_s_48014, midas_s_101, midas_s_54021, Clostridium chartatabidum, midas_s_57157, Clostridium saudiense, midas_s_8895, Clostridium tertium, and midas_s_93503.

[0099] In yet a further specific embodiment of the invention, the composition comprises one or more species of the genus midas_g_12 selected from midas_s_4202, midas_g_12 unclassified, midas_s_3429, midas_s_15546, and midas_s_91465.

[0100] In still another specific embodiment of the invention, the composition comprises one or more species of the genus Ca. Cloacimonas selected from midas_s_301, midas_s_246, Ca. Cloacimonas unclassified, midas_s_3011, and midas_s_110004.

[0101] In still a further specific embodiment of the invention, the composition comprises one or more species of the genus Turicibacter selected from Turicibacter sanguinis and Turicibacter unclassified.

[0102] In a specific embodiment of the invention, the ratio of abundance between the species specified above in the composition is as defined in Table 3. It is understood that each numeric value disclosed in Table 3 includes a variation of ±5% of the stated value.

[0103] Table 3. Ratio of abundance between species.

[0104]

[0105]

[0106]

[0107] In another embodiment of the invention, the composition comprises one or more additional microbe of at least one genus selected from midas_g_467, Ca. Brevefilum, midas_g_30901, Anaerolinea, midas_g_156, Flexilinea, Leptolinea, p-1088-a5 gut group, Ca. Anammoximicrobium, Christensenellaceae R-7 group, Truepera, Syntrophomonas, midas_g_7812, Lachnospiraceae NK3A20 group, Nocardioides, Corynebacterium, DMER64, Mesotoga, Limnobacter, and midas_g_1946.

[0108] In a specific embodiment of the invention, the ranges of each of the additional microbes in the composition are at least 0.1% of the entire population of microbes. In another specific embodiment of the invention, the ratio of abundance of the additional microbes in the composition is as defined in Table 4. It is understood that each numeric value disclosed in Table 4 includes a variation of ±5% of the stated value.

[0109] Table 4. Ratio of abundance of additional microbes.

[0110]

[0111]

[0112] As would be appreciated by a person of skills in the art, the ratios presented in Table 4 are in comparison to the ratios presented in Table 2, namely, the ratio of the genera listed in Table 2 remains unchanged even if the composition comprises one or more of the additional microbes listed in Table 4. Thus, in a non-limiting example, assuming the composition comprises microbes of the 10 genera Romboutsia, Paeniclostridium, Methanobacterium, Methanothrix, Clostridium sensu stricto 1, midas_g_12, Ca. Cloacimonas, Turicibacter, Flexilinea, and Mesotoga, the ratio between the microbes would be between 21:14:3:29:19:19:17:17:1:3 and 43:30:139:125:49:34:176:42:5:18 (Romboutsia:Paeniclostridium:Methanobacterium: Methanothrix Clostridium sensu stricto l:midas_g_12:Ca. Cloacimonas:Turicibacter:Flexilinea: Mesotoga), including a variation of ±5% of the stated value, according to both Tables 2 and 4.In another embodiment of the invention, the combinations of microbes in the compositions as described herein are at a total concentration of at least lxio9cells per 1 gram of volatile solids (gVS) of the composition. In a specific embodiment of the invention, the microbes in the composition are at a total concentration of between lxio9and lxio12cells per gVS of the composition.

[0113] In yet a further embodiment of the invention, the microbes in the compositions of the invention are at a total concentration of between lxio7and 2.4xl012cells per 1 g or per 1 ml of the composition.

[0114] According to an embodiment of the invention, the composition further comprises additives selected from minerals, growth factors, nutrients, buffers, preservatives, and any combination thereof that contribute to the growth of the microbes in the reactor.

[0115] According to another embodiment of the invention, the composition further comprises excipients and / or acceptable carriers, such as cryoprotectants and stabilizers.

[0116] As would be appreciated by a person of skills in the art, the microbes in the compositions of the invention may be the only biomethane producers in the reactor, or may act additively or synergistically with the microbes originally present in the reactor to produce biomethane.

[0117] In some embodiments of the invention, the compositions described herein are used as a starter culture in the reactor in which the anaerobic digestion takes place. In other embodiments of the invention, the compositions are applied to already running reactors to improve the biomethane yield.

[0118] In another aspect, the invention provides a method for enhancing biomethane production by anaerobic digestion of waste, the method comprising:

[0119] a) providing waste into a reactor configured to perform anaerobic digestion;

[0120] b) adding to the reactor a composition that comprises, among other things, at least one microbe of each of the genera Romboutsia, Paeniclostridium, Methanobacterium,Methanothrix, Clostridium sensu stricto 1, midas_g_12, Candidatus Cloacimonas (Ca. Cloacimonas), and Turicibacter, thereby obtaining inoculated waste;

[0121] c) anaerobically incubating the inoculated waste from step (b), thereby generating biogas containing methane; and

[0122] d) collecting the biogas generated in step (c).

[0123] The reactor, according to the present invention, is an anaerobic digester. Examples of suitable reactors include, but are not limited to, batch-flow digesters, semi-continuous flow digesters, continuous flow digesters, continuous stirred tank reactors (CSTRs, also known as complete mix reactors), plug flow digesters, fixed-film digesters, expanded granular sludge bed (EGSB) reactors, up-flow anaerobic sludge blanket (UASB), anaerobic filter, vertical anaerobic sludge blanket (VASB), two-stage digesters, thermophilic digesters, mesophilic digesters, dry fermentation digesters, batch digesters, and landfills.

[0124] In one embodiment of the invention, the waste is livestock waste, municipal waste, and industrial waste. In another embodiment of the invention, the waste is in the form of sludge, liquid waste, or solid waste. In a specific embodiment of the invention, the waste is in the form of sludge.

[0125] The inoculation step comprises adding the compositions of the invention to the reactor containing the waste.

[0126] In one embodiment of the invention, the composition is introduced into the reactor once. In another embodiment of the invention, the composition is added to the reactor continuously, at intervals ranging between once a day and once a month. In a specific embodiment of the invention, the composition is added to the reactor once every two weeks.

[0127] In one embodiment of the invention, each genus of microbes in the composition described in step b) of the method is present in said composition at a range of 0.3% or more of the entire population of microbes. In another embodiment of the invention, each genus of microbes in the composition described in step b) of the method is present in the composition at the ratios defined in Table 2 above.In another embodiment of the invention, the composition added to the reactor in step b) of the method comprises one or more additional microbes of the genera selected from midas_g_467, Ca. Brevefilum, midas_g_30901, Anaerolinea, midas_g_156, Flexilinea, Leptolinea, p-1088-a5 gut group, Ca. Anammoximicrobium, Christensenellaceae R-7 group, Truepera, Syntrophomonas, midas_g_7812, Lachnospiraceae NK3A20 group, Nocardioides, Corynebacterium, DMER64, Mesotoga, Limnobacter, and midas_g_1946. In a specific embodiment of the invention, the ranges of each of the additional microbes in the composition described in step b) of the method are at least 0.1%. In another specific embodiment of the invention, the ratio of abundance of the additional microbes in the composition described in step b) of the method is as defined in Table 4 above.

[0128] In some embodiments of the invention, prior to adding the composition to the reactor, the composition is dissolved in a biological solution. According to one embodiment of the invention, the biological solution is any liquid / buffer solution or semi-liquid solution that maintains the viability of microorganisms. According to another embodiment of the invention, the biological solution is a portion of sludge from the reactor into which the composition is to be added.

[0129] As would be appreciated by a person of skills in the art, the step of adding the composition to the reactor includes, for example, dripping, infusing, or spraying the composition into or onto the waste present in the reactor, each in its own embodiment.

[0130] In one embodiment of the invention, the composition is added to the reactor containing the waste at a composition-to-waste ratio of from 1:10,000 to 1:3 (gVS:gVS). According to another embodiment of the invention, the composition is added to the reactor containing the waste at a ratio of from 1:10,000 to 1:4 (gVS:gVS) composition-to-waste. According to yet another embodiment of the invention, the composition is administered at a ratio of from 1:10,000 to 1:50 (gVS:gVS). In a specific embodiment of the invention, the composition is administered to the waste at a composition-to-waste ratio of 1:10,000 to 1:100 (gVS:gVS). According to a further embodiment of the invention, the step of adding a composition to the reactor comprises feeding the composition to the anaerobic reactor containing the waste at a ratioranging between 0.01% gVS:gVS to 25% gVS:gVS, between 0.01% and 2% gVS:gVS, or between 0.01% and 1% gVS:gVS, each in its own embodiment.

[0131] In another embodiment of the invention, the composition is applied to the reactor containing the waste such that the combinations of microbes as described herein are at a total concentration of at least lxio9cells per gVS of the composition. In a specific embodiment of the invention, the microbes in the composition are at a total concentration of between lxio9and lxio12cells per gVS of the composition.

[0132] In yet a further embodiment of the invention, the composition is applied to the reactor containing the waste such that the combinations of microbes as described herein are at a total concentration of between lxio7and 2.4xl012cells per 1 g or per 1 ml of the composition.

[0133] According to another embodiment of the invention, the composition is applied to the reactor containing the waste at a volume of 1 ml to 2.5 L of the composition per 10 L of waste or per 10 kg of waste.

[0134] The anaerobic incubation in step c) of the method of the invention is the normal operation of the anaerobic digester.

[0135] In one embodiment of the invention, the incubating of step c) of the method is performed at conventional conditions of AD plants, such as standard temperature, pH, salinity, etc.

[0136] In step d) of the method described herein, the biogas is collected in a suitable container, such as a gas holder. In some embodiments, the biogas is in compressed form.

[0137] The biogas collected in step d) of the method of the invention is raw biogas that includes other gases other than methane, such as carbon dioxide (CO2), water vapor (H2O), oxygen (O2), nitrogen (N2), and hydrogen sulfide (H2S). To utilize biomethane, the traces of the other gases produced during the anaerobic digestion should normally be reduced to the minimum possible.Thus, in one embodiment of the invention, the method further comprises the step:

[0138] e) removing gases other than methane from the biogas collected in step d).

[0139] The removal of gases other than methane in step e) of the method can be carried out using standard techniques, such as pressure swing adsorption (PSA), absorption, membrane separation, strippers, scrubbers, and cryogenic separation / distillation. In a non-limiting example, CO2and H2S are removed from the collected biogas by using NaOH scrubbers.

[0140] The invention will now be described with reference to specific examples and materials. The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of specific embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

[0141] EXAMPLE 1

[0142] Determination of biomethane potential of the composition in raw sludge form in municipal wastewater

[0143] Determination of the potential of a composition of the invention in raw sludge form to improve biomethane production of a municipal wastewater sample was carried out on a municipal wastewater sludge sample obtained from an anaerobic reactor of a municipal wastewater treatment plant (WWTP), using a composition comprising the combination of microbes shown in Table 5 below. The composition was sampled from the first anaerobic reactor of a full-scale facility for degrading organic matter (livestock manure) comprising a collecting basin, two anaerobic reactors, and one aerobic reactor in sequential communication with one another, as shown in Fig. 1, after 5 hours of mixing in the reactor.

[0144] Table 5. Ratio of abundance between microbes in the composition

[0145]

[0146]

[0147] Fig. 1 is a schematic representation of an IES system 400 - Integrated Ecosystem Solution by SGTECH, a process and apparatus for treating organic matter (livestock manure). A detailed explanation of systems of this kind can be found, for instance, in PCT Publication No. WO 2020 / 058970 Al and in PCT Publication No. WO 2020 / 058971 Al. The system of this figure is based on a feeding vessel 410 for feeding homogenized and optionally diluted liquid feedstock to a coupled apparatus of anaerobic and aerobic biological processes that include anaerobic digesters 401 and 402, aerobic reactor 403, settling apparatus 404 from which treated water is fed to storage tank 405, and digestate to tank 406. The treated water from storage tank 405 may optionally be used to dilute the feeding vessel 410. The IES system is characterized by recycling communication between the structural elements, as indicated by the solid and dashed arrows. A gas flow meter measures 407 the biogas produced that can be further utilized as a heat and power source of energy, such as generator 409 or injected as methane to the gas grid after a process of gas upgrade at upgrader 408. Organic matter is fed to the system from feeding vessel 410.

[0148] Fig. 2 is a schematic representation of the setup of the BMP test - Biomethane potential laboratory system employed in this Example. This laboratory system, shown at numeral 100, quantifies the potential yield of biogas production from various organic substrates through controlled anaerobic digestion. The system consists of 15 glass bottles indicated by numeral 101, of 500 ml volume each, which are operated autonomously under anaerobic conditions, in a water bath 102, to control the temperature set to 37°C with mixing paddles (not shown). The reactors consist of a total of 400 ml of organic matter, including the added composition in the treatment groups. The biomethane, indicated by arrow 105, is measured by gas flowmeter103 after CO2 is removed by base scrubbers 104. The system measures continuously for up to 25 days in the examples below.

[0149] Fig. 3A shows a schematic representation of a conventional anaerobic digestion system 300 that degrades organic matter (livestock manure) received through arrow 305 into digestate and biogas. The system includes anaerobic digester 301, gas flowmeter 302 to measure the biogas produced that can be further utilized as a heat and power source of energy at generator 303, or injected as methane to the gas grid after a process of gas upgrade at upgrader 304. The resulting digestate may be stored in storage tank 306.

[0150] Fig. 3B shows a schematic representation of the wastewater treatment plant 200 - a facility designed to treat and purify organic matter (wastewater) through physical, chemical, and biological processes, including anaerobic digestion with energy recovery through biogas production. The system includes a primary and secondary clarifiers (not shown), which, through arrow 201, feed organic matter to anaerobic digester 202, gas flowmeter 203 to measure the biogas produced that can be further utilized as a heat and power source of energy, indicated by generator 204, or injected as methane to the gas grid after a process of gas upgrade 205. After dewatering at 206, the sludge is disposed of or utilized for compost.

[0151] As would be appreciated by a person of skills in the art, the BMP setup of Fig. 2 essentially contains the same components as the conventional AD system of Fig. 3A, as well as the conventional wastewater treatment plant of Fig. 3B. Thus, the BMP constitutes an acceptable lab-scale test system to simulate actual large-scale digesters and treatment plants.

[0152] Triplicates of each treatment group were stirred with a magnetic stirrer in a 5 L Erlenmeyer flask at room temperature for 10 minutes and then transferred to reactor bottles. The total weight of the waste samples in each reactor bottle was 400 g. The headspace of each reactor bottle was flushed with nitrogen (N2) gas to ensure anaerobic conditions. Pipes from each bottle were put in a beaker with distilled water, and nitrogen gas was flushed for 10 seconds. During the experiment, the reactor bottles were placed in a water bath at 37 °C. Levels of biomethane were monitored during the 7 days of the experiment using the Aurora software volumetric BioProcess Control, Sweden. Traces of CO2and H2S gases were removed from thebiomethane by filling 100 ml glass bottles with NaOH 3M (base) scrubbers, which were connected to reactor bottles during the entire duration of the experiment.

[0153] The biomethane volume accumulation (in Nml units) was compared between the following four treatment groups:

[0154] (1) 400 g of municipal wastewater sample - 0% of composition added (CO);

[0155] (2) 320 g of municipal wastewater sample with 80 g composition in raw sludge form (C80) - 25% of composition added at a ratio of about 10% gVS:gVS;

[0156] (3) 200 g municipal wastewater sample with 200 g composition in raw sludge form (C200) - 50% composition added at a ratio of about 25% gVS:gVS; and

[0157] (4) 400 g composition in raw sludge form (C400) - 100 % composition.

[0158] Fig. 4 is a graph showing the effect of the composition that includes the combination of 8 genera of microbes as specified in Table 5 above in the form of raw sludge on biomethane production of Municipal Wastewater anaerobic sludge after 7 days of experiment (N=3).

[0159] The results indicate that by increasing the concentration of the composition in the reactor, an increase in biomethane production was observed respectively. The composition alone, compared to untreated municipal waste, showed twice the production of biomethane production. Additionally, as shown in Table 6 below, a decrease in the %VS values of the samples during the experiment was confirmed, indicating that organic matter was successfully decomposed, leading to biogas production.

[0160] Table 6. %VS values at the start (day 0) and end (day 7) of the experiment

[0161]

[0162] EXAMPLE 2Determination of biomethane potential of the composition in capsulated sludge form in municipal wastewater

[0163] Determination of the potential of a composition of the invention in the form of sludge capsules to improve biomethane production of a Municipal Wastewater sample was carried out on a municipal wastewater sludge sample obtained from an anaerobic reactor of the municipal wastewater treatment plant (WWTP). The composition was sampled from the first anaerobic reactor of a full-scale facility for degrading organic matter (livestock manure) comprising a collecting basin, two anaerobic reactors, and one aerobic reactor in sequential communication with one another, as shown in Fig. 1, after 5 hours of mixing in the reactor. A BMP test, as described in Example 1, was employed to quantify the potential yield of biogas production.

[0164] The composition sludge capsules were prepared according to the following illustrative procedure. A quantity of 2.0 L of the sample of raw sludge was collected and kept at 4 °C for up to 5 days. The sludge was shaken, transferred to a suitable Erlenmeyer, and stirred for 5 minutes until a homogeneous solution was obtained. The sludge was then divided into 50 ml test tubes, 40 ml of sludge in each test tube. The samples were vortexed for 10 seconds in order to release microorganisms that may have clung to the remains of straw, stones, mud, dirt, etc., and then passed through a filtering strainer funnel with 1,000 pm (=lmm) pores in order to get rid of residues of straw, stones, mud, and dirt. The liquid filtrates were collected in new 50 ml test tubes and centrifuged in a 4 °C cooled centrifuge, at 2,800 g (4,000 rpm) for 20 minutes. The supernatants were separated from the pellets and discarded. The pellets are the capsulated sludge composition according to an embodiment of the invention. The capsules were kept frozen at -20 °C until use.

[0165] While the examples employed the pellets directly, the skilled person will appreciate that they can alternatively be packaged in biodegradable pouches, compressed into tablet form, or worked into any other convenient form known in the art. Thus, for the sake of brevity, the Examples herein generally refer to the pellets (concentrated sludge) as "capsules" or "capsulated sludge".

[0166] The combination of microbes used in the experiment is shown in Table 7 below.Table 7. Ratio of abundance between microbes in the composition

[0167]

[0168] Triplicates of each treatment group were stirred with a magnetic stirrer in a 5 L Erlenmeyer flask at room temperature for 10 minutes and then transferred to reactor bottles. The total weight of the waste samples in each reactor bottle was 400 g. The headspace of each reactor bottle was flushed with nitrogen (N2) gas to ensure anaerobic conditions. Pipes from each bottle were put in a beaker with distilled water, and nitrogen gas was flushed for 10 seconds. During the experiment, the reactor bottles were placed in a water bath at 37 °C. Levels of biomethane were monitored during the 20 days of the experiment using the Aurora software volumetric BioProcess Control, Sweden. Traces of CO2and H2S gases were removed from the biomethane by filling 100 ml glass bottles with NaOH 3M (base) scrubbers, which were connected to reactor bottles during the entire duration of the experiment.

[0169] The biomethane volume accumulation (in Nml units) was compared between the following three treatment groups:

[0170] (1) 400 g of municipal wastewater sample - no composition added (0 cap);

[0171] (2) 395 g of municipal wastewater sample with 2 composition capsules, which is equivalent to about ~5 g composition added at a ratio of about 10% gVS:gVS (2 cap); and

[0172] (3) 388 g municipal wastewater sample with 5 composition capsules, which is equivalent to about 12 g composition added at a ratio of about 25% gVS:gVS (5 cap).Fig. 5 shows the effect of the composition that includes the combination of 8 genera of microbes as specified in Table 7 above in the form of sludge capsules on biomethane production of Municipal Wastewater anaerobic sludge after 20 days of experiment ( N=3).

[0173] As shown in Fig. 5, an increase in the concentration of the composition in the reactor led to an increase in biomethane production. Additionally, as shown in Table 8 below, a decrease in the %VS values of the samples during the experiment was confirmed, indicating that organic matter was successfully decomposed, leading to biogas production. These results strengthen the results obtained in the experiment detailed in Example 1 and illustrate the improvement in methane production obtained by adding the composition of the invention.

[0174] Table 8. %VS values at the start (day 0) and end (day 20) of the experiment

[0175]

[0176] EXAMPLE 3

[0177] Determination of biomethane potential of the composition in capsulated sludge form in livestock waste

[0178] Determination of the potential of a composition of the invention in the form of sludge capsules to improve biomethane production of a livestock waste sample was carried out on a livestock waste sludge sample obtained from a conventional anaerobic digestion system. The composition was sampled from the first anaerobic reactor of a full-scale facility for degrading organic matter (livestock manure) comprising a collecting basin, two anaerobic reactors, and one aerobic reactor in sequential communication with one another, as shown in Fig. 1, after 5 hours of mixing in the reactor. A BMP test, as described in Example 1, was employed to quantify the potential yield of biogas production.

[0179] The composition sludge capsules were prepared according to the procedure described in Example 2.The combination of microbes used in the experiment is shown in Table 9 below.

[0180] Table 9. Ratio of abundance between microbes in the composition

[0181]

[0182] Triplicates of each treatment group were stirred with a magnetic stirrer in a 5 L Erlenmeyer flask at room temperature for 10 minutes and then transferred to a reactor bottle. The total weight of the waste samples in each reactor bottle was 400 g. The headspace of each reactor bottle was flushed with nitrogen (N2) gas to ensure anaerobic conditions. Pipes from each bottle were put in a beaker with distilled water, and nitrogen gas was flushed for 10 seconds. During the experiment, the reactor bottles were placed in a water bath at 37 °C. Levels of biomethane were monitored during the 18 days of the experiment using the Aurora software volumetric BioProcess Control, Sweden. Traces of CO2and H2S gases were removed from the biomethane by filling 100 ml glass bottles with NaOH 3M (base) scrubbers, which were connected to reactor bottles during the entire duration of the experiment.

[0183] The biomethane volume accumulation (in Nml units) was compared between the following three treatment groups:

[0184] (1) 400 g of livestock waste sample - no composition added (0 cap);

[0185] (2) 395 g of livestock waste sample with 2 composition capsules, which is equivalent to about ~5 g composition added at a ratio of about 10% gVS:gVS (2 cap); and

[0186] (3) 388 g livestock waste sample with 5 composition capsules, which is equivalent to about 12 g composition added at a ratio of about 25% gVS:gVS (5 cap).Fig. 6 shows the effect of the composition comprising a combination that includes the 8 genera of microbes as specified in Table 9 above, in the form of sludge capsules, on biomethane production of livestock waste anaerobic sludge after 18 days of experiment (N=3).

[0187] As shown in Fig. 6, an increase in the concentration of the composition in the reactor led to an increase in biomethane production. Additionally, as shown in Table 10 below, a decrease in the %VS values of the samples during the experiment was confirmed, indicating that organic matter was successfully decomposed, leading to biogas production. These results strengthen the results obtained in the experiments detailed in Examples 1 and 2 and illustrate the improvement in methane production obtained by adding the composition of the invention.

[0188] Table 10. %VS values at the start (day 0) and end (day 18) of the experiment

[0189]

[0190] EXAMPLE 4

[0191] Determination of biomethane potential of the composition in capsulated sludge form in livestock waste in a small-scale CSTR system

[0192] Determination of the potential of a composition of the invention in the form of sludge capsules to improve biomethane production of a livestock waste sample in a conventional anaerobic digestion system (small-scale CSTR) was carried out using two CSTRs containing the livestock waste, as shown in Fig. 7, one of them treated with a composition sampled from the first anaerobic reactor of a full-scale facility for degrading organic matter (livestock manure) comprising a collecting basin, two anaerobic reactors, and one aerobic reactor in sequential communication with one another, as shown in Fig. 1, after 5 hours of mixing in the IES reactor.Fig. 7 is a schematic representation of the setup for this experiment. The system shown at numeral 500 includes two anaerobic digesters 501 and 502, each having a mixing rotor (not shown), and a shared feeding vessel 503 from which livestock waste is fed to both digesters 501 and 502. The system further includes an additive dosing port 504 from which the composition is fed to anaerobic digester 501. A gas flowmeter 505 measures the biogas produced by anaerobic digester 501, and a gas flowmeter 506 measures the biogas produced by anaerobic digester 502.

[0193] Each digester contains 27 liters of livestock waste and is operated under anaerobic conditions, at 37 °C. Gas flow rate and content were measured 5 days a week, and biomethane values were calculated per liter of waste to determine the effect of the composition administration on biomethane production.

[0194] After completion of the setup and acclimation phase, 15 capsules of the composition (roughly equivalent to 38 g) were administered to anaerobic reactor 501 on Day 1 at a ratio of 1% gVS:gVS (C15), with a subsequent dose applied after two weeks (Day 15). Thus, the composition was administered to the waste at a ratio of 1% gVS per administration. Anaerobic reactor 502 functioned as untreated control (UTC).

[0195] The composition sludge capsules were prepared according to the procedure described in Example 2.

[0196] The combination of microbes used in the experiment is shown in Table 11 below.

[0197] Table 11. Ratio of abundance between microbes in the composition

[0198]

[0199]

[0200] The results are shown in Table 12 and Fig. 8.

[0201] Table 12. Change in biomethane flow rate over time

[0202]

[0203] It is observed that in the days following each administration of the composition, the levels of biomethane flow rate in the composition-treated reactor were significantly higher than the biomethane flow rate in the untreated reactor. Additionally, although the untreated waste showed some fluctuations in the flow rate levels, the flow rate levels of biomethane in the composition-treated reactor remained relatively stable and mostly higher than those of the untreated waste. Thus, these results show that the overall treatment with the composition of the invention has increased biomethane production from livestock waste in the CSTR experiment setup. It is expected that increasing the frequency of administration of the composition will improve biomethane production even more.

[0204] These results strengthen the results of the BMP tests shown in Examples 1-3 and further support the advantages of using the composition of the invention for enhancing methane production during anaerobic digestion of waste.

Claims

CLAIMS1. A composition for enhancing biomethane production by anaerobic digestion of waste, wherein the composition comprises at least one microbe of each of the genera: Romboutsia, Paeniclostridium, Methanobacterium, Methanothrix, Clostridium sensu stricto 1, midas_g_12, Candidatus Cloacimonas (Ca. Cloacimonas), and Turicibacter.

2. The composition according to claim 1, wherein:a) the at least one microbe of the genus Romboutsia comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1-221; and / orb) the at least one microbe of the genus Paeniclostridium comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 222-339; and / orc) the at least one microbe of the genus Methanobacterium comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 340-478; and / ord) the at least one microbe of the genus Methanothrix comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 479-919; and / ore) the at least one microbe of the genus Clostridium sensu stricto 1 comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 920-1319; and / orf) the at least one microbe of the genus midas_g_12 comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1320-1455; and / org) the at least one microbe of the genus Ca. Cloacimonas comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1456-1755; and / orh) the at least one microbe of the genus Turicibacter comprises a 16S rRNA encoding gene comprising a V4 region sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1756-2024.

3. The composition according to claim 1 or 2, wherein the microbes of each genus are present in the composition at the following ratios:

4. The composition according to any one of claims 1-3, wherein the composition comprises one or more of the species midas_s_34, midas_s_5918, Romboutsia unclassified, midas_s_31016, midas_s_43100, midas_s_59815, Romboutsia lituseburensis, Paeniclostridium unclassified, Paeniclostridium sordellii, Methanobacterium unclassified, midas_s_3006, Methanobacterium formicicum, Methanobacterium lacus, midas_s_9526, Methanothrix_soehngenii, Methanothrix_harundinacea, midas_s_36, Methanothrix unclassified, Methanothrix thermoacetophila, midas_s_94826, midas_s_90667, midas_s_64, Clostridium sensu stricto 1 unclassified, midas_s_3795, Clostridium disporicum, Clostridium butyricum, midas_s_48014, midas_s_101, midas_s_54021, Clostridium chartatabidum, midas_s_57157, Clostridium saudiense, midas_s_8895,Clostridium tertium, midas_s_93503, midas_s_4202, midas_g_12 unclassified, midas_s_3429, midas_s_15546, midas_s_91465, midas_s_301, midas_s_246, Ca. Cloacimonas unclassified, midas_s_3011, midas_s_110004, Turicibacter sanguinis, and Turicibacter unclassified.

5. The composition according to claim 4, wherein the species are present in the composition at the following ratios:

6. The composition according to any one of claims 1-5, wherein the composition comprises one or more additional microbe of the genus selected from midas_g_467, Ca. Brevefilum, midas_g_30901, Anaerolinea, midas_g_156, Flexilinea, Leptolinea, p-1088-a5 gut group, Ca. Anammoximicrobium, Christensenellaceae R-7 group, Truepera, Syntrophomonas, midas_g_7812, Lachnospiraceae NK3A20 group, Nocardioides, Corynebacterium, DMER64, Mesotoga, Limnobacter, and midas_g_1946.

7. The composition according to claim 6, wherein the one or more additional microbe is present in the composition at the following ratios:

8. The composition according to any one of claims 1-7, wherein the composition comprises at least a total of lxio9microbial cells per gVS of the composition.

9. The composition according to claim 8, wherein the composition comprises a total of lxio9to lxio12microbial cells pergVS of the composition.

10. The composition according to any one of claims 1-7, wherein the composition comprises a total concentration of microbial cells of from lxio7to 2.4xl012cells per 1 g or per 1 ml of the composition.

11. The composition of any one of claims 1-10, wherein the composition further comprises additives selected from minerals, growth factors, nutrients, buffers, preservatives, and any combination thereof, that contribute to the growth of the microbes.

12. A method for enhancing biomethane production by anaerobic digestion of waste, the method comprising:a) providing waste into a reactor configured to perform anaerobic digestion;b) adding a composition according to any one of claims 1-11 to the reactor, thereby obtaining inoculated waste;c) anaerobically incubating the inoculated waste from step (b), thereby generating biogas containing methane; andd) collecting said biogas generated in step (c).

13. The method according to claim 12, wherein the waste is in the form of sludge.

14. The method according to claim 12 or 13, wherein the composition is added to the reactor once.

15. The method according to claim 12 or 13, wherein the composition is added to the reactor at intervals ranging between once a day and once a month.

16. The method according to claim 15, wherein the composition is added to the reactor once every two weeks.

17. The method according to any one of claims 12-16, wherein prior to adding the composition to the reactor, the composition is dissolved in a biological solution.

18. The method according to any one of claims 12-17, wherein the composition is added to the reactor at a composition-to-waste ratio of from 0.01% to 25% (gVS:gVS).

19. The method according to any one of claims 12-18, wherein the composition is applied to the reactor containing the waste at a volume of 1 ml to 2.5 L per 10 L of waste or at a volume of 1 ml to 2.5 L per 10 kg of waste.

20. The method according to any one of claims 12-19, wherein the method further comprises:e) removing gases other than methane from the biogas collected in step d).

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