Methods, devices, and systems for producing trihalomethane

Abstract

The present disclosure provides a method for producing trihalomethane, comprising: adding at least one vanadium-dependent haloperoxidase (VHPO) enzyme to a solution in an reactor; adding one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV to the solution; adding hydrogen peroxide to the solution; and separating one or more trihalomethane produced by the at least one VHPO enzyme from the solution. The present disclosure also provides a device for producing trihalomethane that implements the method for producing trihalomethane.

Description

[0001] METHODS, DEVICES, AND SYSTEMS FOR PRODUCING TRIHALOMETHANE

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 711,407, filed on October 24, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0005] A Sequence Listing in XML format, submitted under 37 C.F.R. §§ 1.831-1.835, entitled 3000157-005000 Sequence Listing. xml, 13,827 bytes in size, generated on October 22, 2024, and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is incorporated by reference into the specification for its disclosures.

[0006] FIELD

[0007] The present disclosure relates to devices, methods, and processes associated with the use of at least one vanadium-dependent haloperoxidase (VHPO) enzyme to produce trihalomethanes which, when administered to ruminants, can reduce the methane production in the rumen, thereby reducing methane emissions.

[0008] BACKGROUND

[0009] In the realm of livestock management, one of the significant challenges is the control of methane emissions. Ruminants, such as cows, sheep, and goats, are known to produce methane as a byproduct of their digestive process. This methane is released into the atmosphere, contributing to greenhouse gas emissions and global warming. Furthermore, the production of methane also represents a loss of energy for the animal, as the energy contained in the methane could have been used for growth or milk production.

[0010] Marine macroalgae, seaweeds, are producers of halogenated natural products. Biosynthesis of halogenated molecules is linked to reactive oxygen species (ROS) such as hydrogen peroxide that are substrates for haloperoxidase enzymes that promote the formation of halogenated molecules. It has been shown that vanadium-dependent haloperoxidases (VHPOs), halogenating enzymes that are present in seaweeds, are involved in bromoform biosynthesis. The importance of bromoform synthesis is that it can inhibit methane production. The antimethanogenic activity of bromoform was studied in vitro by isolating bioactive compounds from the red seaweed Asparagopsis.

[0011] The red macroalga Asparagopsis taxiformis has been linked to the reduction of methane (CH4) from beef cattle by up to 99%. A recent study shows that A. taxiformis is a highly efficient feed supplement for CH4 mitigation during enteric fermentation. The antimethanogenic compounds found in A. taxiformis are: bromoform, dibromochloromethane, bromochloroacetic acid, dibromoacetic acid and dichloromethane. However, bromoform, a trihalomethane, is the most abundant antimethanogenic compound found in A. taxiformis. Bromoform and dibromochloromethane had the highest activity at inhibiting methane production.

[0012] Bromoform is a trihalomethane that has been found to interfere with the methanogenesis pathway by serving as a competitive inhibitor or analog of methyl-coenzyme M reductase (MCR), preventing the final catalysis step.

[0013] It would be useful, practically and commercially, to be able to reduce the methane emissions produced by ruminants, and in particular, domestic livestock, by using a source of bromoform that is easily produced with limited impact on the environment. Fungal strains expressing VHPOs present an alternative to marine macroalgae for accomplishing this objective. To facilitate the goal, it would be attractive to develop devices and methods suitable for large scale production of bromoform utilizing the catalysis activity of VHPO enzymes.

[0014] SUMMARY

[0015] In some embodiments, the present disclosure relates to a method for producing trihalomethane, comprising: adding at least one vanadium-dependent haloperoxidase (VHPO) enzyme to a solution, for example a solution in an reactor; adding one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV to the solution

[0016]

[0017] Formula III Formula IV adding hydrogen peroxide to the solution; and separating one or more trihalomethane produced by the at least one VHPO enzyme from the solution.

[0018] In some embodiments, wherein in Formula II,

[0019] R3, R4, Rs, Rs, R7, and Rs each independently represents hydrogen, halogen, hydroxyl, carboxyl, substituted or unsubstituted Ci-Ce alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, .scc-butyl, tert-butyl, or combinations thereof, wherein one or more methylene groups of the substituted or unsubstituted Ci-Ce alkyl of R3, R4, Rs, Rs, R7, and / or Rs may optionally be replaced by -O-, -NH-, -S-, -COO-, -COS-, CONH- , -CSNH-, and / or -CSO-.

[0020] In some embodiments, wherein the compound of Formula II is one or more of the following:

[0021]

[0022] In some embodiments, wherein the compound of Formula II is Formula Il-d. In some embodiments, wherein the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV and the hydrogen peroxide are pre-mixed to form a mixture, the mixture is subsequently fed to the reactor.

[0023] In some embodiments, at least one pH buffering agent is added to the solution. Nonlimiting examples of the at least one pH buffering agent include: formic acid, acetic acid, propanoic acid, carbonic acid, fumaric acid, benzoic acid, oxalic acid, malonic acid, maleic acid, succinic acid, lactic acid, gluconic acid, adipic acid, o-toluic acid, benzene tetracarboxylic acid, citric acid, isocitric acid, aconitic acid, propane- 1,2, 3 -tricarboxylic acid, itaconic acid, citraconic acid, hydrogen chloride (HC1), sulfuric acid (H2SO4), nitric acid (HNO3), phosphoric acid (H3PO4), sodium hydroxide (NaOH), potassium hydroxide (KOH), monopotassium phosphate (KH2PO4), dipotassium phosphate (K2HPO4), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), borate, sulfamic acid (H3NSO3), monosodium phosphate (NaH2PO4), disodium phosphate (Na2HPO4), sodium chloride (NaCl), potassium chloride (KC1), calcium chloride (CaCh), magnesium chloride (MgCb), ammonia, [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 7V,7V-Bis(2- hydroxyethyl)glycine (Bicine), tris(hydroxymethyl)aminomethane (Tris), / V-[ l ,3-Dihydroxy-2- (hydroxymethyl)propan-2-yl]glycine (Tricine), 3-[[l,3-dihydroxy-2-(hydroxymethyl)propan-2- yl] amino] -2-hydroxypropane-l -sulfonic acid (TAPSO), 2-[4-(2-Hydroxyethyl)piperazin-l- yl] ethane- 1 -sulfonic acid (HEPES), 2-{[l,3-Dihydroxy-2-(hydroxymethyl)propan-2- yl]amino} ethane- 1 -sulfonic acid (TES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine- / / , M -bis(2-ethanesulfonic acid) (PIPES), and 2-( / V-morpholino)ethanesulfonic acid (MES).

[0024] In some embodiments, the at least one pH buffering agent is added to the mixture.

[0025] In some embodiments, the method further comprising: adding one or more halide salt to the solution. Non-limiting examples of the one or more halide salt include: potassium chloride (KC1), potassium bromide (KBr), potassium iodide (KI), sodium chloride (NaCl), sodium bromide (NaBr), and sodium iodide (Nal).

[0026] In some embodiments, the one or more halide salt is added to the mixture.

[0027] In some embodiments, the at least one VHPO enzyme is immobilized and / or confined to the reactor.

[0028] In some embodiments, wherein immobilization and / or confinement of the at least one VHPO enzyme to the reactor is accomplished by: coupling or ataching the at least one VHPO enzyme to a wall of the reactor, coupling or ataching the at least one VHPO enzyme to a plurality of beads, and / or confining the at least one VHPO enzyme with an ultrafiltration membrane.

[0029] In some embodiments, the at least one VHPO enzyme is coupled or attached to the wall of the reactor via covalent binding, affinity binding, and / or physical adsorption.

[0030] In some embodiments, the at least one VHPO enzyme is coupled or attached to the plurality of beads via covalent binding, affinity binding, and / or physical adsorption.

[0031] In some embodiments, the plurality of beads are made of a material comprising silica, magnetic materials, resins, polymers, and / or noble metals.

[0032] In some embodiments, the ultrafiltration membrane is made of a material comprising cellulose, hemicellulose, cellulose acetate, regenerated cellulose, polyether sulfone (PES), polysulfone (PSU), polyphenylene sulfone (PPSU), poly(bisphenol-A sulfone) (PSF), poly(arylene sulfone) (PAS), polylactide, poly(aryl ether ether ketone) (PEEK), polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET), polyethylene chlorotrifluoroethylene (PECTFE), polyethylene tetrafluoroethylene (PETFE), polytetrafluoroethylene (PTFE), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), polyvinylidene fluoride (PVDF), thermoplastic polyurethane (TPU), polyvinyl-fluoride, ethylenechlorotrifluoroethylene copolymer, ethylene-tetrafluoroethylene-copolymer, fluoroethylenepropylene copolymer, perfluoralkoxy polymer, and / or polychloro-trifluoroethylene.

[0033] In some embodiments, the ultrafiltration membrane is installed in an ultrafiltration cassete.

[0034] In some embodiments, the at least one VHPO enzyme, the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV, and the hydrogen peroxide are blended in the solution in the reactor.

[0035] In some embodiments, the solution is filtered through the ultrafiltration cassette.

[0036] In some embodiments, the solution is blended by an agitator blender, a paddle mixer, a dual shaft blender, a ribbon blender, a rotary blender, a vibrational blender, a static mixer, or a vertical blender.

[0037] In some embodiments, the method further comprising pumping an air flow into the solution. In some embodiments, the air flow is directed to pass through the solution, such that the one or more trihalomethane produced by the at least one VHPO enzyme is separated from the solution and volatilized by the air flow.

[0038] In some embodiments, the method further comprising directing the air flow to a tank, the tank contains one or more high surface area material to capture the one or more trihalomethane.

[0039] In some embodiments, the tank is a rotating drum.

[0040] In some embodiments, the one or more high surface area material has a surface area of about 50 to about 5000 m2 / g, about 100 to about 5000 m2 / g, about 200 to about 5000 m2 / g, about 300 to about 5000 m2 / g, about 500 to about 5000 m2 / g, about 500 to about 4000 m2 / g, about 500 to about 3000 m2 / g, about 750 to about 3000 m2 / g, about 900 to about 3000 m2 / g, about 1000 to about 3000 m2 / g, or about 500 to about 1500 m2 / g. In other embodiments, the high surface area material has a surface area of 50 to 5000 m2 / g, 100 to 5000 m2 / g, 200 to 5000 m2 / g, 300 to 5000 m2 / g, 500 to 5000 m2 / g, 500 to 4000 m2 / g, 500 to 3000 m2 / g, 750 to 3000 m2 / g, 900 to 3000 m2 / g, 1000 to 3000 m2 / g, or 500 to 1500 m2 / g.

[0041] In some embodiments, the one or more high surface area material is a carbonaceous material, a hyper-crosslinked polymer, an aerogel, and / or a porous sorbent. Non-limiting examples of the carbonaceous material include: activated carbon (activated charcoal), graphene, graphene oxide, and derivatives thereof. In some embodiments, the one or more high surface area material is activated carbon.

[0042] In some embodiments, the method further comprising: after directing the air flow to pass through the solution, directing the air flow to pass through at least one desiccant or water absorbing material. Non-limiting examples of the at least one desiccant or water absorbing material include: activated alumina, calcium chloride (CaCb), calcium oxide (CaO), calcium hydride (CaHz), calcium sulfate (CaSOfl, cobalt(II) chloride (C0CI2), copper(II) sulfate (CuSOfl, lithium chloride (LiCl), lithium bromide (LiBr), magnesium chloride (MgCh), magnesium sulfate (MgSOfl, magnesium perchlorate (Mg(C104)2), Nafion, phosphorus pentoxide (P4O10), potassium carbonate (K2CO3), potassium hydroxide (KOH), sodium hydroxide (NaOH), silica gel, sodium, sodium chlorate (NaQOs), sodium chloride (NaCl), sodium sulfate (Na2SO4), sucrose, sulfuric acid (H2SO4), triethylene glycol, and a combination thereof.

[0043] In some embodiments, the method further comprising extracting the one or more trihalomethane from the one or more high surface area material. In some embodiments, a hydrophobic solvent is applied to extract the one or more trihalomethane.

[0044] In some embodiments, wherein the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV, and / or the hydrogen peroxide are continuously added to the solution. In some embodiments, wherein the one or more halide salt is continuously added to the solution.

[0045] In some embodiments, the method further comprising: adding one or more alkaline earth metal salt to the solution. Non-limiting examples of the one or more alkaline earth metal salt include: calcium oxide (CaO), calcium acetate, calcium carbonate (CaCO,), calcium chloride (CaCh), calcium citrate, calcium gluconate, calcium sulfate (CaSC ), calcium bromide (CaBn), calcium hydroxide (Ca(OH)2), calcium nitrate (Ca(NOs)2), magnesium oxide (MgO), magnesium acetate, magnesium carbonate (MgCCh), magnesium chloride (MgCb), magnesium citrate, magnesium gluconate, magnesium sulfate (MgSC ), magnesium bromide (MgBn), magnesium hydroxide (Mg(0H)2), magnesium nitrate (Mg(NOs)2), and / or a combination thereof.

[0046] In some embodiments, wherein the at least one VHPO enzyme is recombinantly expressed by a fungal strain. Non-limiting examples of the genera to which the fungal strain belongs include Arxula, Candida, Ogataea, Kluyveromyces, Pichia, Saccharomyces, and Yarrowia. Within these genera, the fungal strain belongs to any one of the following non-limiting species: Saccharomyces cerevisiae, Yarrowia lipolytica, or Pichia pastoris.

[0047] In some embodiments, the fungal strain is a filamentous fungus. Additional genera to which the fungal strain belongs include Altemaria, Ascomycota, Bipolaris, Cadophora, Cladorrhinum, Clohesyomyces, Curvularia, Darksidea, Didymella, Dothideomycetes, Drechslera, Exserohilum, Epicoccum, Exophiala, Gaeumannomyces, Gliomastix, Helotiales, Keissleriella, Laburnicola, Lachnum, Leptodontidium, Magnaporthiopsis, Mollisia, Neocamarosporium, Oidiodendron, Ophiosphaerella, Penicillium, Phaeosphaeria, Phialophora, Phoma, Pleosporales, Poaceascoma, Septoriella, Slopeiomyces, Sordariales, Trametes, Trematosphaeria, Trichoderma, and Tricladium. Within these genera, the fungal strain belongs to any one of the following non-limiting species: Altemaria chlamydospora, Cladorrhinum foecundissimum, Curvularia inaequalis, Curvularia spicifera, Curvularia intermedia, Curvularia pseudobrachyspora, Curvularia hawaiiensis, Curvularia protuberata, Curvularia lunata, Darksidea alpha, Darksidea zeta, Leptodontidium orchidicola, Magnaporthiopsis panicorum, Mollisia cinerea, Neocamarosporium phragmitis, Oidiodendron citrinum, Penicillium penarojense, Penicillium griseolum, Phaeosphaeria caricis, Phaeosphaeria microscopica, Phaeosphaeria poagena, Phaeosphaeria sinensis, Poaceascoma lochia, Septoriella leuchtmannii, Slopeiomyces cylindrosporus, Trametes versicolor, Trematosphaeria hydrela, Trematosphaeria terricola, Trichoderma lixii, and Tricladium terrestre.

[0048] In some embodiments, the VHPO has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0049] In some embodiments, the VHPO has an amino acid sequence of SEQ ID NO: 1. In some embodiments, the VHPO has an amino acid sequence of SEQ ID NO: 2. In some embodiments, the VHPO has an amino acid sequence of SEQ ID NO: 3. In some embodiments, the VHPO has an amino acid sequence of SEQ ID NO: 4. In some embodiments, the VHPO has an amino acid sequence of SEQ ID NO: 5. In some embodiments, the VHPO has an amino acid sequence of SEQ ID NO: 6. In some embodiments, the VHPO has an amino acid sequence of SEQ ID NO: 7. In some embodiments, the VHPO has an amino acid sequence of SEQ ID NO: 8. In some embodiments, the VHPO has an amino acid sequence of SEQ ID NO: 9.

[0050] In some embodiments, the trihalomethane is selected from the group consisting of chloroform bromoform, iodoform, bromodichloromethane, dibromochloromethane and combinations thereof. In some embodiments, the trihalomethane is bromoform.

[0051] In some embodiments, wherein the fungal strain is Curvularia sp. 4388 (NMI Accession No. V22 / 011149), Curvularia lunata US-991 (ATCC Accession No. PTA-127504), Curvularia pseudobrachyspora US-215 (ATCC Accession No. PTA-127509), Curvularia spicifera US-188 (ATCC Accession No. PTA-127510), Curvularia inaequalis US-998 (ATCC Accession No. PTA- 127511), Curvularia intermedia US-429 (ATCC Accession No. PTA-127512), Curvularia hawaiiensis US-446 (ATCC Accession No. PTA-127601), Curvularia protuberata US-1111 (ATCC Accession No. PTA-127607), or a mutant thereof having all identifying characteristics of the strain.

[0052] In some embodiments, the solution has a pH value of between about 4.0 to about 10.0, between about 4.0 to about 9.5, between about 4.0 to about 9.0, between about 4.0 to about 8.5, between about 4.0 to about 8.0, between about 4.0 to about 7.5, between about 4.0 to about 7.0, between about 4.0 to about 6.75, between about 4.0 to about 6.5, between about 4.25 to about 6.5, between about 4.5 to about 6.5, between about 4.75 to about 6.5, between about 5.0 to about 6.5, between about 5.0 to about 6.25, or between about 5.0 to about 6.0.

[0053] In some embodiments, the present disclosure relates to a device for producing trihalomethane, comprising: a reactor, wherein a solution and at least one vanadium-dependent haloperoxidase (VHPO) enzyme are disposed in the reactor; a first reservoir, the first reservoir is connected to the reactor and contains one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV a second reservoir, the second reservoir is connected to the reactor and contains hydrogen peroxide; and a tank, the tank is connected to the reactor and contains one or more high surface area material.

[0054] In some embodiments, the first reservoir transfers the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV to the reactor; and the second reservoir transfers the hydrogen peroxide to the reactor. In some embodiments, the first reservoir further contains one or more halide salt.

[0055] In some embodiments, the device further comprising: a third reservoir, the third reservoir is connected to the first reservoir, the second reservoir, and the reactor, wherein the third reservoir receives the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV transferred from the first reservoir and the hydrogen peroxide transferred from the second reservoir, such that the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV and the hydrogen peroxide are pre-mixed to form a mixture, wherein the third reservoir transfers the mixture to the reactor.

[0056] In some embodiments, the device further comprising: a fourth reservoir, the fourth reservoir is connected to the reactor and contains at least one pH buffering agent, wherein the fourth reservoir transfers the at least one pH buffering agent to the reactor.

[0057] In some embodiments, the fourth reservoir transfers the at least one pH buffering agent to the third reservoir.

[0058] In some embodiments, the device further comprising: an air pump, the air pump pumps an air flow into the solution.

[0059] In some embodiments, the device further comprising: a fifth reservoir, the fifth reservoir is connected to the reactor and the tank, the fifth reservoir contains at least one desiccant or water absorbing material, wherein the air flow is directed to flow from the reactor to the tank through the fifth reservoir, such that a moisture contained with the air flow is removed by the at least one desiccant or water absorbing material.

[0060] In some embodiments, wherein one or more trihalomethane is produced by the at least one VHPO enzyme in the reactor, and the one or more trihalomethane is captured by the one or more high surface area material in the tank.

[0061] In some embodiments, the device further comprising: an ultrafiltration cassette, the ultrafiltration cassette is connected to the reactor and contains an ultrafiltration membrane to confine the at least one VHPO enzyme in the reactor, wherein the solution flows from the reactor to the ultrafiltration cassette such that the solution is filtered through the ultrafiltration membrane, and the solution flows from ultrafiltration cassette back to the reactor.

[0062] In some embodiments, the device further comprising a sixth reservoir, the sixth reservoir is connected to the reactor and contains one or more alkaline earth metal salt.

[0063] In some embodiments, the first reservoir, the second reservoir, the third reservoir, the fourth reservoir, and / or the sixth reservoir continuously transfer the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV, the hydrogen peroxide, the at least one pH buffering agent, the one or more halide salt, and / or the one or more alkaline earth metal salt to the reactor.

[0064] In some embodiments, the reactor comprises an agitator blender, a paddle mixer, a dual shaft blender, a ribbon blender, a rotary blender, a vibrational blender, a static mixer, and / or a vertical blender, and wherein the solution is blended in the reactor.

[0065] In some embodiments, the device further comprises a first pump. In some embodiments, transfer of the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV and / or the one or more halide salt from the first reservoir to the reactor is facilitated by the first pump.

[0066] In some embodiments, the device further comprises a second pump. In some embodiments, transfer of the hydrogen peroxide from the second reservoir to the reactor is facilitated by the second pump.

[0067] In some embodiments, the device further comprises a third pump. In some embodiments, transfer of the solution from the reactor to the ultrafiltration cassette and return from the ultrafiltration cassette to the reactor is facilitated by the third pump.

[0068] In some embodiments, the device further comprises a fourth pump. In some embodiments, transfer of the at least one pH buffering agent from the fourth reservoir to the reactor is facilitated by the fourth pump.

[0069] In some embodiments, the present disclosure relates to a device for producing trihalomethane, comprising: a reactor, wherein a solution and at least one vanadium-dependent haloperoxidase (VHPO) enzyme are disposed in the reactor; a first reservoir, the first reservoir contains one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV

[0070] Formula III Formula IV a second reservoir, the second reservoir contains hydrogen peroxide; a third reservoir; a fourth reservoir, the fourth reservoir contains at least one pH buffering agent, wherein the third reservoir is connected to the first reservoir, the second reservoir, the fourth reservoir, and the reactor, wherein the first reservoir transfers the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV to the third reservoir, the second reservoir transfers the hydrogen peroxide to the third reservoir, and the fourth reservoir transfers the at least one pH buffering agent to the third reservoir, such that the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV, the hydrogen peroxide, and the at least one pH buffering agent are pre-mixed to form a mixture, wherein the third reservoir transfers the mixture to the reactor; an air pump, the air pump pumps an air flow into the solution; an ultrafiltration cassette, the ultrafiltration cassette is connected to the reactor and contains an ultrafiltration membrane to confine the at least one VHPO enzyme in the reactor, wherein the solution flows from the reactor to the ultrafiltration cassette such that the solution is filtered through the ultrafiltration membrane, and the solution flows from ultrafiltration cassette back to the reactor; and a tank, the tank is connected to the reactor and contains one or more high surface area material, wherein the air flow is directed to flow from the reactor to the tank, wherein one or more trihalomethane is produced by the at least one VHPO enzyme in the reactor, wherein the one or more trihalomethane is captured by the one or more high surface area material in the tank.

[0071] In some embodiments, the present disclosure relates to a method for reducing methane emissions from a ruminant. In some embodiments, the ruminant is Bos taurus or Bos indicus. In some embodiments, the ruminant is Ovis aries or Capra hircus.

[0072] The following description is intended only by way of example, and simply illustrates certain selected embodiments of devices, systems, and processes that are consistent with the disclosed subject matter as claimed herein.

[0073] BRIEF DESCRIPTION OF THE DRAWINGS

[0074] FIG. 1 depicts an exemplary device for producing trihalomethane according to the present invention.

[0075] FIG. 2 depicts another exemplary device for producing trihalomethane according to the present invention.

[0076] FIG. 3 depicts yet another exemplary device for producing trihalomethane according to the present invention.

[0077] FIG. 4 depicts a further exemplary device for producing trihalomethane according to the present invention.

[0078] FIG. 5 depicts an alternative exemplary device for producing trihalomethane according to the present invention.

[0079] FIG. 6 depicts another alternative exemplary device for producing trihalomethane according to the present invention. FIG. 7 depicts the VHPO reaction mechanism.

[0080] FIG. 8 depicts the readings from a pH pump and a balance during equilibration of the bromoform reaction in a reactor.

[0081] FIG. 9 depicts the readings from a reagent pump and a balance pump during equilibration of the bromoform reaction in a reactor.

[0082] DETAILED DESCRIPTION

[0083] Preferred features, embodiments and variations of the invention may be discerned from the following detailed description which provides sufficient information for those skilled in the art to perform the invention. The detailed description is not to be regarded as limiting the scope of the preceding summary of the invention in any way.

[0084] In compliance with the statute, the invention has been described in language more or less specific to structural or methodical features. The term “comprises” and its variations, such as “comprising” and “comprised of’ is used throughout in an inclusive sense and not to the exclusion of any additional features. It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims appropriately interpreted by those skilled in the art.

[0085] Throughout the specification and claims (if present), unless the context requires otherwise, the term “substantially” or “about” will be understood to not be limited to the value for the range qualified by the terms.

[0086] It is to be understood that unless specifically stated otherwise, references to “a,” “an,” and / or “the” may include one or more than one and that reference to an item in the singular may also include the item in the plural. Reference to an element by the indefinite article “a,” “an” and / or “the” does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements. As used herein, the term “comprise,” and conjugations or any other variation thereof, are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. A “ruminant” is a mammal of the order Artiodactyla that digests plant-based food by initially softening and partially fermenting it within the animal's first stomach chambers, then regurgitating the semi-digested mass, now known as cud, and chewing it again. The process of rechewing the cud to further break down plant matter and stimulate digestion is called “ruminating”. Ruminants have a digestive tract with four chambers, namely the rumen, reticulum, omasum and abomasum. In the first two chambers, the rumen and the reticulum, the food is mixed with saliva and separates into layers of solid and liquid material. Solids clump together to form the cud, or bolus. The cud is then regurgitated, chewed slowly to completely mix it with saliva, which further breaks down fibers. Fiber, especially cellulose, is broken down into glucose in these chambers by symbiotic anaerobic bacteria, protozoa and fungi. The broken-down fiber, which is now in the liquid part of the contents, then passes through the rumen into the next stomach chamber, the omasum. The food in the abomasum is digested much like it would be in the monogastric stomach. Digested gut contents are finally sent to the small intestine, where the absorption of the nutrients occurs. Almost all the glucose produced by the breaking down of cellulose is used by the symbiotic bacteria. Ruminants get their energy from the volatile short chain fatty acids (VFAs) produced by the bacteria, namely acetate, propionate, butyrate, valerate, and isovalerate. Ruminants included cattle, goats, sheep, giraffes, yaks, deer, antelope, and others.

[0087] As used herein, the term “bovid” includes any member of the family Bovidae, which include hoofed mammals such as antelope, sheep, goats, and cattle, among others.

[0088] As used herein, the term “reducing” includes the reduction of amount of substance in comparison with a reference. For example, the reduction in the amount of total gas and / or methane produced by a ruminant animal or animals administered a composition comprising a fungal strain according to the present invention, relative to an animal or animals not administered a composition comprising a fungal strain of the present invention. The reduction can be measured in vitro with an artificial rumen system that simulates anaerobic fermentation, or in vivo with animals confined in respiration chambers. It is within the knowledge and skill of those trained in the art to assess enteric methanogenesis by a ruminant animal.

[0089] As used herein, the term “reducing methane production” refers to the reduction of methane produced in the gastro-intestinal tract. The term includes the specific volume of methane generated as a result of anaerobic fermentation, for example, in the systems described herein. Fermentation in the rumen and the gut of a ruminant gives rise to production of methane. The present invention aims to reduce this process, such as to reduce the total amount of methane produced in the gastrointestinal tract. It is within the knowledge and skill of those trained in the art to assess methane production by a ruminant animal.

[0090] The term “specific activity” as used herein is defined as the units of activity in a given amount of protein. Thus, the specific activity is not directly measured but is calculated by dividing 1) the activity in units / ml of the enzyme sample by 2) the concentration of protein in that sample, so the specific activity is expressed as units / mg, where an enzyme unit is defined as moles of product formed / minute. The specific activity of a sample of pure, fully active enzyme is a characteristic of that enzyme. The specific activity of a sample of a mixture of proteins is a measure of the relative fraction of protein in that sample that is composed of the active enzyme of interest.

[0091] The terms “kcat” and “KM” are known to those skilled in the art and are described in Enzyme Structure and Mechanism, 2nd ed. (Ferst; W.H. Freeman Press, NY, 1985; pp 98-120). KM, the Michaelis constant, is the concentration of substrate that leads to half-maximal velocity. The term “kcat”, often called the “turnover number”, is defined as the maximum number of substrate molecules converted to products per active site per unit time, or the number of times the enzyme turns over per unit time. kcat=Vmax / [E], where [E] is the enzyme concentration (Ferst, supra). The terms “total turnover” and “total turnover number” are used herein to refer to the amount of product formed by the reaction of an enzyme with substrate.

[0092] The term “catalytic efficiency” is defined as the kcat / KM of an enzyme. Catalytic efficiency is used to quantify the specificity of an enzyme for a substrate.

[0093] The term “affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (for example, a VHPO enzyme) and its binding partner (for example, a substrate). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, such as, for example, immunoblot, ELISA KD, KinEx A, spectroscopic assays, isothermal titration calorimetry (ITC), biolayer interferometry (BLI), or surface plasmon resonance (SPR) devices. The terms “KD,” “Kd,” “Kd” or “Kd value” as used interchangeably to refer to the equilibrium dissociation constant of an enzyme- substrate interaction.

[0094] The term “binds” to an enzyme or receptor is a term that is well understood in the art, and methods to determine such binding are also well known in the art. A molecule is said to exhibit “binding” if it reacts, associates with, or has affinity for a particular cell or substance and the reaction, association, or affinity is detectable by one or more methods known in the art, such as, for example, immunoblot, ELISA KD, KinEx A, spectroscopic assays, isothermal titration calorimetry (ITC), biolayer interferometry (BLI), surface plasmon resonance (SPR) devices, or etc.

[0095] A “variant” refers to a biologically active polypeptide having at least about 50% amino acid sequence identity with the native sequence polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, deleted, at the N or C-terminus of the polypeptide.

[0096] In some embodiments, a variant or variants described herein have at least about 50% amino acid sequence identity, at least about 60% amino acid sequence identity, at least about 65% amino acid sequence identity, at least about 70% amino acid sequence identity, at least about 75% amino acid sequence identity, at least about 80% amino acid sequence identity, at least about 85% amino acid sequence identity, at least about 90% amino acid sequence identity, at least about 95% amino acid sequence identity, at least about 96% amino acid sequence identity, at least about 97% amino acid sequence identity, at least about 98% amino acid sequence identity, at least about 99% amino acid sequence identity with the native sequence polypeptide.

[0097] Proteins comprising or consisting of an amino acid sequence “at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical”, “at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical”, or similar recitations, to a reference sequence may comprise mutations such as deletions, insertions and / or substitutions compared to the reference sequence. The reference sequence may be, as non-limiting examples, a wild type sequence, a mature wild type sequence, a native sequence, a truncated wild type sequence, a truncated mature wild type sequence, a truncated native sequence, or a sequence disclosed herein. The reference sequence may be, as nonlimiting examples, a wild type sequence, a mature wild type sequence, or a native sequence. In the case of substitutions, the protein consisting of an amino acid sequence at least or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence may correspond to a homologous sequence derived from another species than the reference sequence.

[0098] As used herein, “percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide, or protein sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. A comparison of sequences and determination of percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the identity between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison. D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Addison Wesley). Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALINE™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of sequences being compared. For example, the percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mai. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For example, the NEEDLE program from the EMBOSS package may be used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, Longden, and Bleasby, Trends in Genetics 16, (6) 276-277, emboss.bioinformatics.nl / ). For amino acid sequences, EBLOSUM62 may be used for the substitution matrix. For nucleotide sequence, EDNAFULL may be used. The optional parameters used may be a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

[0099] A “point mutation” is a mutation that involves a single amino acid residue. The mutation may be the loss of an amino acid, substitution of one amino acid residue for another, or the insertion of an additional amino acid residue. An amino acid substitution may include but is not limited to the replacement of one amino acid in a polypeptide with another amino acid.

[0100] An “amino acid substitution” refers to the replacement of one amino acid in a polypeptide with another amino acid. Non-limiting exemplary amino acid substitutions are shown in Table 1. Amino acid substitutions may be introduced into an enzyme of interest and the products screened for a desired activity, for example, retained / improved substrate binding.

[0101] Table 1. Amino acid substitutions may be conservative or non-conservative. In some embodiments, substitutions may be conservative substitutions, in which one amino acid is substituted for another amino acid with similar structural and / or chemical properties.

[0102] Conservative substitutions may comprise those, which are described by Dayhoff in “The Atlas of Protein Sequence and Structure. Vol. 5”, Natl. Biomedical Research, the contents of which are incorporated by reference in their entirety. Amino acids may be grouped according to common side-chain properties:

[0103] (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

[0104] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0105] (3) acidic: Asp, Glu;

[0106] (4) basic: His, Lys, Arg;

[0107] (5) residues that influence chain orientation: Gly, Pro;

[0108] (6) aromatic: Trp, Tyr, Phe.

[0109] Conservative substitutions may entail exchanging a member of one of these classes with a member of the same class. Non-conservative substitutions may entail exchanging a member of one of these classes with a member of another class.

[0110] Conservative substitutions may be made in accordance with Table 2. Methods for predicting tolerance to protein modification may be found in, for example, Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the contents of which are incorporated by reference in their entirety.

[0111] Table 2. Conservative Amino Acid substitution

[0112] Conservative Amino Acid Substitutions

[0113] Amino Acid Substitutions (others are known in the art)

[0114] Ala Ser, Gly, Cys

[0115] Arg Lys, Gin, His

[0116] Asn Gin, His, Glu, Asp

[0117] Asp Glu, Asn, Gin

[0118] Cys Ser, Met, Thr

[0119] Gin Asn, Lys, Glu, Asp, Arg

[0120] Glu Asp, Asn, Gin

[0121] Gly Pro, Ala, Ser

[0122] His Asn, Gin, Lys

[0123] He Leu, Vai, Met, Ala

[0124] Leu He, Vai, Met, Ala

[0125] Lys Arg, Gin, His

[0126] Met Leu, He, Vai, Ala, Phe

[0127] Phe Met, Leu, Tyr, Trp, His

[0128] Ser Thr, Cys, Ala

[0129] Thr Ser, Vai, Ala

[0130] Trp Tyr, Phe

[0131] Tyr Trp, Phe, His

[0132] Vai He, Leu, Met, Ala, Thr

[0133] An “amino acid derivative,” as used herein, refers to any amino acid, modified amino acid, and / or amino acid analogue, that is not one of the 20 common natural amino acids found in humans. Exemplary amino acid derivatives include natural amino acids not found in humans (e.g., seleno cysteine and pyrrolysine, which may be found in some microorganisms) and unnatural amino acids. Exemplary amino acid derivatives, include, but are not limited to, amino acid derivatives commercially available through chemical product manufacturers (e.g., sigmaaldrich.com / chemistry / chemistry-products. html?TablePage=l 6274965, accessed on May 6, 2017, which is incorporated herein by reference). One or more amino acid derivatives may be incorporated into a polypeptide at a specific location using a translation system that utilizes host cells, orthogonal aminoacyl-tRNA synthetases derived from eubacterial synthetases, orthogonal tRNAs, and an amino acid derivative. For further descriptions, see, e.g., U.S. Patent No. 9,624,485.

[0134] In some embodiments, a polypeptide comprises an amino acid substitution with an amino acid derivative. In some embodiments, the amino acid derivative is an alanine derivative, a cysteine derivative, an aspartic acid derivative, a glutamic acid derivative, a phenylalanine derivative, a glycine derivative, a histidine derivative, an isoleucine derivative, a lysine derivative, a leucine derivative, a methionine derivative, an asparagine derivative, a proline derivative, a glutamine derivative, an arginine derivative, a serine derivative, a threonine derivative, a valine derivative, a tryptophan derivative, or a tyrosine derivative.

[0135] Fungal Strains Expressing a Vanadium-Dependent Haloperoxidase

[0136] In certain aspects, the present disclosure provides a method for reducing methane emissions from a ruminant comprising administering to the ruminant a composition comprising a fungal strain, biomass from the fungal strain, a culture supernatant from the fungal strain, or a combination thereof, wherein the fungal strain comprises a vanadium-dependent haloperoxidase (VHPO).

[0137] In some aspects, the amino acid sequence of the VHPO comprises an amino sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. (see Table 3). In one aspect, the VHPO comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0138] In other aspects, the amino acid sequence of the VHPO comprises an amino sequence having a tyrosine at position 69, a tyrosine at position 356, a phenylalanine at position 289, or combinations thereof. In one aspect, the VHPO comprises an amino sequence having a tyrosine at position 69, a tyrosine at position 356, and a phenylalanine at position 289.

[0139] In other aspects, the VHPO is a variant protein of the protein comprising or consisting of the ammo acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. Variant proteins are within the scope of the invention as long as the resulting variant protein retains similar characteristics when compared to the parent peptide. Exemplary modifications are for example conservative substitutions that will result in VHPO variants with similar characteristics to those of the parent molecules. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids may be divided into four families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. Alternatively, the amino acid repertoire may be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally grouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur- containing (cysteine and methionine) (Stryer (ed.), Biochemistry, 2nd ed, WH Freeman and Co., 1981). Non-conservative substitutions can be made to the VHPO variants that involve substitutions of amino acid residues between different classes of amino acids to improve properties of the VHPO variants. Whether a change in the amino acid sequence of a polypeptide or fragment thereof results in a functional homolog can be readily determined by assessing the ability of the modified polypeptide or fragment to produce a response in a fashion similar to the unmodified polypeptide or fragment using the assays described herein. Peptides, polypeptides or proteins in which more than one replacement takes place can readily be tested in the same manner.

[0140] In one aspect, the VHPO comprises an amino sequence having an aromatic amino acid at position 69. In another aspect, the VHPO comprises an amino sequence having a sulfur-containing amino acid at position 69. In another aspect, the VHPO comprises an amino sequence having a basic amino acid at position 69.

[0141] In one aspect, the VHPO comprises an amino sequence having an aliphatic-hydroxyl amino acid at position 72.

[0142] In another aspect, the VHPO comprises an amino sequence having a non-polar amino acid at position 92. In one aspect, the non-polar amino acid at position 92 is proline. In another aspect, the non-polar amino acid at position 92 is leucine.

[0143] In one aspect, the VHPO comprises an amino sequence having an aliphatic-hydroxyl amino acid at position 118. In another aspect, the VHPO comprises an amino sequence having a nonpolar amino acid at position 118. In one aspect, the VHPO comprises an amino sequence having an aliphatic amino acid at position 138. In one aspect, the aliphatic amino acid at position 138 is glycine. In another aspect, the aliphatic amino acid at position 138 is alanine.

[0144] In one aspect, the VHPO comprises an amino sequence having an aliphatic-hydroxyl amino acid at position 260. In another aspect, the VHPO comprises an amino sequence having a nonpolar amino acid at position 260.

[0145] In one aspect, VHPO comprises an amino sequence having an aliphatic-hydroxyl amino acid at position 536. In one aspect, the aliphatic-hydroxyl amino acid at position 536 is threonine. In one aspect, the aliphatic-hydroxyl amino acid at position 536 is serine.

[0146] In one aspect, VHPO comprises an amino sequence having a basic amino acid at position 569. In one aspect, the basic amino acid at position 569 is lysine. In another aspect, the basic amino acid at position 569 is arginine.

[0147] In one aspect, VHPO comprises an amino sequence having an aliphatic amino acid at position 588. In one aspect, the aliphatic amino acid at position 588 is valine. In another aspect, the aliphatic amino acid at position 588 is isoleucine.

[0148] In another aspect, the VHPO comprises an amino sequence having a non-polar amino acid at position 592. In one aspect, the non-polar amino acid at position 592 is proline. In another aspect, the non-polar amino acid at position 592 is leucine.

[0149] In one aspect, the VHPO comprises an amino sequence having a basic amino acid at position 594. In another aspect, the VHPO comprises an amino sequence having an acidic amino acid at position 594. In another aspect, the VHPO comprises an amino sequence having an uncharged polar amino acid at position 594.

[0150] In another aspect, the VHPO comprises an amino sequence having a non-polar amino acid at position 606.

[0151] Attorney Docket No.: LOAM-B009-01WO

[0152] Table 3. VHPO amino acid sequences of fungal strains and consensus sequence.

[0153] Atorney Docket No.: LOAM-B009-01WO

[0154] Atorney Docket No.: LOAM-B009-01WO

[0155] Atorney Docket No.: LOAM-B009-01WO

[0156] Attorney Docket No.: LOAM-B009-01WO

[0157] * Messerschmidt A, Wever R. X-ray structure of a vanadium-containing enzyme: chloroperoxidase from the fungus Curvularia inaequalis. Proc Natl Acad Sci U S A. 1996 Jan 9;93(l):392-6. doi: 10.1073 / pnas.93.1.392. PMID: 8552646; PMCID: PMC40244.

[0158] Keto-Enol Tautomerism of Ketones and Diketones

[0159] Ketones are organic compounds having the structure R-C(=O)-R’. Diketones are organic compounds having two ketone groups. Structures of exemplary diketones are shown below.

[0160] 1,2-Diketone 1,3-Diketone 1 ,4-Diketone

[0161] (B

[0162] Ketone groups may be converted into alkenol (enol) groups spontaneously via tautomerization, the keto-enol tautomerism, which is a chemical equilibrium between a keto form and an enol form. Diketones may also undergo similar tautomerism, transforming into enol tautomers. Keto-enol tautomerism may be affected by different variables and / or conditions. For example, differences in solvent, pH value, and / or temperature may drive the equilibrium toward different directions. Both intermolecular and intramolecular interactions can play a significant role here in determining the keto-enol composition at thermodynamic equilibrium. Exemplary ketoenol tautomerisms of ketones and diketones are shown below.

[0163] Keto-enol tautomerism of 3-Pentanone:

[0164] Keto-enol tautomerism of Dimedone:

[0165] Keto-enol tautomerism of Acetylacetone: Keto-enol tautomerism of Triacetic acid lactone:

[0166] It has been reported that for dimedone, both keto and enol forms co-exist when dissolved in chloroform; whereas in DSMO, only enol form exists. Richard J. Cremlyn et al., NMR spectral studies of dimedone-aldehyde adducts Part 1. 'H and13C NMR spectral studies of dimedone, 52 Spectrochim. Acta A 1423-1432 (1996); Poulomi Majumdar et al., Chemistry of Dimedone for Synthesis of Oxygen-, Nitrogen-, and Sulfur- Containing Heterocycles from 2-(3-Hydroxy-5,5- dimethylcyclohex-2-enylidene)malononitrile, 43 Synth. Commun. 899-914 (2013). The contents of each of these references are herein incorporated by reference in their entireties.

[0167] Inventors have found that, for dimedone, only enol form exists when dissolved in methanol; and for triacetic acid lactone, only enol form exists when dissolved in DMSO. It was not clear, from the literature, whether triacetic acid lactone could exist in keto form.

[0168] Halogenation of the Substrates and Production of Trihalomethanes

[0169] VHPO enzymes catalyze the formation of organo-halogens in the presence of H2O2 and halide anions (fluoride, iodide, chloride and bromide). The catalytic mechanism of VHPOs can be divided into two different reactions (FIG. 7). Reaction 1 corresponds to the formation of the hypohalide HOBr. One of hydroxy groups of the vanadium(V)-dioxo-dihydroxo abstracts a proton from a hydrogen peroxide molecule, which then nucleophilic attacks the vanadium (V) forming a vanadium(V)-dioxo-hydroxo-hydroperoxo-water six-coordinated intermediate. Then, a water is displaced to form a five-coordinated vanadium(V)- dioxo-hydroperoxo intermediate. Finally, the hydroxy from the hydroperoxo is transferred to Br-, forming the hypohalide, HOBr, while a proton is transferred to the vanadium complex to reinstate the initial vanadium(V)-dioxo-dihydroxo species (Mubarak et al. 2020). Reaction 2 describes the formation of the halogenated compound. Despite all efforts, the full reaction mechanism has not yet been determined at the QM and QM / MM level (Gerard et al. 2023). Moreover, the substrate pocket is still unknown.

[0170] Organic substrates can be halogenated by VHPO enzymes, which involves oxidizing the substrate with the addition of one or more halogen. Certain substrates, such as triacetic acid lactone, can be further broken down, eventually producing trihalomethanes such as bromoform. A possible reaction mechanism is depicted below.

[0171] Organic Substrates

[0172] Various ketones and diketones are suitable to be applied as organic substrates for the present invention. For example, one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV may be applied as organic substrates for the present invention:

[0173] Formula III Formula IV

[0174] Formula I

[0175] In Formula I, Ri and R2 each independently represents hydroxyl, carboxyl, substituted or unsubstituted Ci-C& alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, .scc-butyl, tertbutyl, or combinations thereof. In some embodiments, one or more methylene groups of the substituted or unsubstituted Ci-C& alkyl of Ri and / or R2 may optionally be replaced by -O-, -NH-, -S-, -COO-, -COS-, C0NH-, -CSNH-, and / or -CSO-. In some embodiments, the compound of Formula I comprises one or more of the following:

[0176] . In some embodiments, the compound of Formula I is Formula La, Formula Lh, Formula

[0177] LI, Formula I-j, Formula I-k, and / or Formula 1-1.

[0178] Formula II

[0179] In Formula II, R3, R4, R5, R6, R7, and R8 each independently represents hydrogen, halogen, hydroxyl, carboxyl, substituted or unsubstituted Ci-Ce alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or combinations thereof. In some embodiments, one or more methylene groups of the substituted or unsubstituted Ci-Ce alkyl of R3, R4, Rs, Rs, R7, and / or Rs may optionally be replaced by -O-, -NFL, -S-, -COO-, -COS-, C0NH-, -CSNH-, and / or -CSO-. In some embodiments, the compound of Formula II is one or more of the following:

[0180]

[0181] . In some embodiments, the compound of Formula II is Formula Il-d. Formula III

[0182] In Formula III, R9 and Rio each independently represents hydrogen, halogen, carboxyl, substituted or unsubstituted Ci-C& alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or combinations thereof. In some embodiments, one or more methylene groups of the substituted or unsubstituted Ci-C& alkyl of R9 and / or Rio may optionally be replaced by -O-, -NH-, -S-, -COO-, -COS-, C0NH-, -CSNH-, and / or -CSO-. In some embodiments, the compound of Formula III is Formula IILa:

[0183] Formula IILa

[0184] Formula IV

[0185] In Formula IV, Rn and R12 each independently represents hydrogen, carboxyl, substituted or unsubstituted Ci-Ce alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or combinations thereof. In some embodiments, one or more methylene groups of the substituted or unsubstituted Ci-C& alkyl of Rn and / or R12 may optionally be replaced by -O-, -NH- , -S-, -COO-, -COS-, C0NH-, -CSNH-, and / or -CSO-. In some embodiments, the compound of Formula IV is Formula IV-a:

[0186] Formula IV-a

[0187] Deposited Fungal Strains

[0188] Biological deposits of each of the fungal strains listed in Table 4 were made on the dates shown at the American Type Culture Collection (ATCC®), located at 10801 University Blvd., Manassas, VA 20110, USA, or the National Measurement Institute (NMI), 1 / 153 Bertie Street, Port Melbourne, Victoria 3207, Australia, under the provisions of the Budapest Treaty and assigned by each International Depositary Authority (IDA) the accession numbers indicated. Upon issuance of a patent, all restrictions upon the deposits will be irrevocably removed. The deposits are intended to meet the requirements of 37 CFR §§ 1.801-1.809. The deposits will be maintained in the ID As for a period of 30 years, or 5 years after the last request, or for the effective, enforceable life of the patent, whichever is longer, and will be replaced, if necessary, during that period; and the requirements of 37 CFR §§ 1.801-1.809 are met.

[0189] Table 4. Fungal strains deposited with the ATCC and NMI International Depositary Authorities.

[0190] Immobilization and / or Confinement of the VHPO Enzymes

[0191] The VHPO enzymes are placed in the reactor, where the halogenation reaction takes place, along with the reaction solution. To improve the yield of the trihalomethanes and to reduce the loss of active VHPO enzymes, the VHPO enzymes are restricted from leaving the reactor. This may be done by immobilizing and / or confining the VHPO enzymes in the reactor. For example, the VHPO enzymes may be coupled or attached to the wall of the reactor. The coupling or attaching may be done by various techniques known in the art. For example, the VHPO enzymes may be covalently coupled to the wall by: (1) activating the surface of the wall using linker molecules such as glutaraldehyde or carbodiimide, and (2) coupling the VHPO enzymes with the linker molecules via the enzyme functional groups such as amino groups, carboxylic groups, phenolic groups, sulfhydryl groups, thiol groups, imidazole groups, indole groups, and / or hydroxyl groups. Alternatively, the VHPO enzymes may be coupled or attached to the wall by affinity binding and / or physical adsorption. In this manner, the VHPO enzymes may be immobilized and / or confined within the reactor.

[0192] Similarly, the VHPO enzymes may also be coupled or attached to a plurality of beads that have a higher density and thus, less likely to be removed from the reactor. Techniques used to couple or attach the VHPO enzymes to the plurality of beads are similar to those used in coupling / attaching to the wall. Thus, as previously described, the VHPO enzymes may be coupled or attached to the plurality of beads by covalent binding, affinity binding, and / or physical adsorption.

[0193] In addition, an ultrafiltration membrane may be used to filter the reaction solution, such that during removal of the unwanted by-products, the VHPO enzymes may be confined within the reactor by the ultrafiltration membrane. In this way, the loss of active VHPO enzymes may be further reduced. A more detailed description of the use of ultrafiltration membranes is provided below.

[0194] High Surface Area Materials

[0195] High surface area materials are materials with high surface area. Typically, high surface area materials have a surface area of at least 50 m2 / g, as measured by the BET method (Brunauer- Emmett-Teller adsorption method). The high surface area materials applied in the present invention typically have a surface area of about 50 to about 5000 m2 / g, about 100 to about 5000 m2 / g, about 200 to about 5000 m2 / g, about 300 to about 5000 m2 / g, about 500 to about 5000 m2 / g, about 500 to about 4000 m2 / g, about 500 to about 3000 m2 / g, about 750 to about 3000 m2 / g, about 900 to about 3000 m2 / g, about 1000 to about 3000 m2 / g, or about 500 to about 1500 m2 / g. Details of each species of the high surface area materials are further described below. Activated Carbon

[0196] Activated carbons are complex products which are difficult to classify on the basis of their behaviour, surface characteristics and other fundamental criteria. However, a broad classification is made for general purposes based on their size, preparation methods, and industrial applications.

[0197] Powdered Activated Carbon (PAC)

[0198] Normally, activated carbons (R 1) are made in particulate form as powders or fine granules less than 1.0 mm in size with an average diameter between 0.15 and 0.25 mm. Thus, they present a large surface to volume ratio with a small diffusion distance. Activated carbon (R 1) is defined as the activated carbon particles retained on a 50-mesh sieve (0.297 mm).

[0199] Powdered activated carbon (PAC) material is finer material. PAC is made up of crushed or ground carbon particles, 95-100% of which will pass through a designated mesh sieve. The ASTM classifies particles passing through an 80-mesh sieve (0.177 mm) and smaller as PAC. It is not common to use PAC in a dedicated vessel, due to the high head loss that would occur. Instead, PAC is generally added directly to other process units, such as raw water intakes, rapid mix basins, clarifiers, and gravity filters.

[0200] Granular Activated Carbon (GAC)

[0201] Granular activated carbon (GAC) has a relatively larger particle size compared to powdered activated carbon and consequently presents a smaller external surface. Diffusion of the adsorbate is thus an important factor. Granular activated carbon are suitable for adsorption of gases and vapours because gaseous substances diffuse rapidly. Granulated activated carbons are used for air filtration and water treatment, as well as for general deodorization and separation of components in flow systems and in rapid mix basins. GAC can be obtained in either granular or extruded form. GAC is designated by sizes such as 8x20, 12x40, 20x40, or 8x30 for liquid phase applications and 4x6, 4x8 or 4x10 for vapor phase applications. A 20x40 carbon is made of particles that will pass through a U.S. Standard Mesh Size No. 20 sieve (0.84 mm) (generally specified as 85% passing) but be retained on a U.S. Standard Mesh Size No. 40 sieve (0.42 mm) (generally specified as 95% retained). AWW A (1992) B604 uses the 50-mesh sieve (0.297 mm) as the minimum GAC size. The most popular aqueous-phase carbons are the 12x40 and 8x30 sizes because they have a good balance of size, surface area, and head loss characteristics. As opposed to powders, granules may have a lower propensity to produce dust during processing (e.g., feed manufacturing and delivery).

[0202] Extruded Activated Carbon (EAC)

[0203] Extruded activated carbon (EAC) combines powdered activated carbon with a binder, which are fused together and extruded into a cylindrical shaped activated carbon block with diameters from 0.8 to 130 mm. These are mainly used for gas phase applications because of their low pressure drop, high mechanical strength and low dust content.

[0204] Bead Activated Carbon (BAC)

[0205] Bead activated carbon (BAC) is made from petroleum pitch and supplied in diameters from approximately 0.35 to 0.80 mm. Like EAC, it is also noted for its low pressure drop, high mechanical strength and low dust content, but with a smaller grain size. Its spherical shape makes it preferred for fluidized bed applications such as water filtration.

[0206] Impregnated Carbon

[0207] Porous carbons containing several types of inorganic impregnates such as iodine and silver are impregnated carbon. Cations such as aluminium, manganese, zinc, iron, lithium, and calcium have also been prepared for specific applications. Due to its antimicrobial and antiseptic properties, silver loaded activated carbon is used as an adsorbent for purification of domestic water. Drinking water can be obtained from natural water by treating the natural water with a mixture of activated carbon and aluminium hydroxide (Al(0H)3), a flocculating agent. Impregnated carbons are also used for the adsorption of hydrogen sulphide (H2S) and thiols.

[0208] Polymer Coated Carbon

[0209] This is a process by which a porous carbon can be coated with a biocompatible polymer to give a smooth and permeable coat without blocking the pores. The resulting carbon is useful for various applications including hemoperfusion. Woven Carbon

[0210] There is a technology of processing technical rayon fiber into activated carbon cloth for carbon filtering. Adsorption capacity of activated cloth is greater than that of activated charcoal (BET theory) surface area: 500-1500 m2 / g, pore volume: 0.3-0.8 cm3 / g). Owing to the different forms of activated material, it can be used in a wide range of applications.

[0211] Carbon Nanotubes (CNTs)

[0212] CNTs are cylindrical structures made of carbon atoms arranged in a hexagonal lattice. They have an extremely high surface area and exceptional mechanical, thermal, and electrical properties. CNTs find applications in fields like electronics, materials science, and nanotechnology.

[0213] Graphene

[0214] Graphene is a single layer of carbon atoms arranged in a hexagonal lattice. It has an extremely high surface area and remarkable electronic and thermal conductivity. Graphene is used in various industries, including electronics, energy storage, and composite materials.

[0215] Carbon Aerogels

[0216] Carbon aerogels are lightweight, highly porous materials with a three-dimensional network structure. They are used in applications such as energy storage devices (supercapacitors), catalysis, and as lightweight structural materials.

[0217] Carbon Black

[0218] Carbon black is a fine powder produced by the incomplete combustion of hydrocarbons. It is widely used as a reinforcing filler in rubber products, such as tires, and as a pigment in inks and coatings.

[0219] Carbon Fibers

[0220] Carbon fibers are composed of carbon atoms bonded together in a crystalline structure. They are known for their high tensile strength, low weight, and resistance to heat. Carbon fibers are used in aerospace, automotive, and sporting goods industries to make lightweight and strong composite materials. Carbon Nanofibers (CNFs)

[0221] Similar to carbon nanotubes, carbon nanofibers are cylindrical structures with diameters in the nanometer range. They are used in various applications, including reinforcement in composites, energy storage devices, and catalyst supports.

[0222] Carbon Cloth and Carbon Paper

[0223] These materials are often made from carbon fibers and are used as electrodes in fuel cells and other energy storage devices. They provide a high surface area for electrochemical reactions.

[0224] Carbon Foam

[0225] Carbon foam is a three-dimensional, open-cell structure made from carbon. It is lightweight and possesses good thermal and electrical conductivity. Carbon foam finds applications in heat exchangers, thermal management systems, and as an electrode material.

[0226] Hyper-Crosslinked Polymers

[0227] Polymeric Resins with Aromatic Rings

[0228] Polymeric adsorbents containing aromatic rings, such as those based on polystyrene- divinylbenzene (PS / DVB) copolymers, may be used for the adsorption of haloforms. The aromatic nature of these resins enhances their affinity for these compounds.

[0229] Poly dimethylsiloxane (PDMS) and Silica-Based Polymers

[0230] PDMS is a silicone-based polymer with high affinity for haloforms. PDMS-coated fibers or particles, as well as other silica-based polymers, may be used in solid-phase microextraction (SPME) for the extraction haloforms.

[0231] Polyacrylate Resins

[0232] Polyacrylate resins are compatible with haloform compounds. They can be tailored to have specific functional groups that enhance their affinity for haloforms. Functional groups of interest include groups containing halides, groups with amide-containing ligands, groups with polar moieties such as hydroxyl and carbonyl groups, and ion exchange groups such as quaternary ammonium and sulfonic groups. Each of these functional groups can enhance the formation of favorable interactions with haloforms.

[0233] Graphene Oxide and Modified Graphene-Based Materials

[0234] Graphene oxide and modified graphene materials may be used for haloform adsorption due to their high surface area and unique properties. Functionalization of graphene can be done to improve selectivity for haloforms.

[0235] Graphene oxide (GO) and reduced graphene oxide (rGO) can be functionalized with oxygen-containing groups (e.g., hydroxyl, carboxyl) on the basal plane and edges. These groups can enhance interactions with haloforms through hydrogen bonding.

[0236] Introduction of halide-containing functional groups (e.g., chloromethyl or chlorophenyl groups) onto the graphene surface can enhance haloform binding.

[0237] Nitrogen-doping of graphene introduces basic nitrogen functionalities, which interact favorably with haloforms. Nitrogen-containing groups can enhance the overall adsorption capacity of the graphene.

[0238] Optimization of the graphene structure can maximizes TT-TT stacking interactions with haloforms. This involves controlling the number of graphene layers or introducing aromatic moieties.

[0239] Incorporation of functionalized carbon nanotubes into graphene-based materials can increase their affinity for haloforms. The tubular structure and functional groups on carbon nanotubes can contribute to improved haloform adsorption.

[0240] Poly(N-vinyl-2-pyrrolidone) (PVP)

[0241] PVP and PVP-based materials are known for their compatibility with a variety of organic compounds. These materials can be used in various forms, such as films or particles, for adsorption applications with haloforms.

[0242] Styrene-Divinylbenzene Polymers

[0243] Styrene-divinylbenzene (Styrene-DVB) copolymers with a macroporous structure can also be used for the adsorption of haloforms. These macroporous structures are characterized by pores with diameters typically in the range of 50 nm to several micrometers. Amberchrom resins, such as Amberchrom CGI 61 C, are macroporous Styrene-DVB copolymers that are commonly used for chromatographic separations. These resins have a high surface area and porosity.

[0244] Chromabond resins, including Chromabond HR-X, are macroporous copolymers of styrene and divinylbenzene. They are utilized in solid-phase extraction (SPE) for sample preparation and purification.

[0245] XAD resins, such as XAD-4 and XAD-16, are macroporous Styrene-DVB copolymers widely used for adsorption and extraction of organic compounds from air and water samples.

[0246] Diaion HP (Hypercrosslinked Porous) resins are macroporous Styrene-DVB copolymers manufactured by Mitsubishi Chemical. They are designed for applications such as chromatography and separation processes.

[0247] NeviPure offers macroporous hyper-cross-linked polystyrene-divinylbenzene resins for various applications, including the removal of impurities from pharmaceutical intermediates.

[0248] MN200 is a macroporous Styrene-DVB copolymer resin used for adsorption applications, including solid-phase extraction (SPE) and chromatography.

[0249] In certain aspects, the hyper-crosslinked polymer comprises a styrene-divinylbenzene group with a macroporous structure. In one aspect, the hyper-crosslinked polymer is selected from the group consisting of PUROSORB® PAD400, PAD500, PAD600, PAD900, PAD1200, PAD350, PAD610, PAD910, PAD950, PAD950C, Amberlite® FPX66, FPX68, Amberlite® XAD2, XAD4, XAD16, XAD1 180, XAD200, XAD2010, XAD16N, XAD1600N, XAD18, XAD1 180N, XAD7HP, DIADION® Sepabe, XAD761 ® HP20, HP20SS, HP21 , SP70, SP700, SP825L, SP850, CHP20, CHP50, SP207, HP2MGL, LEWATIT® AF 5, SEPLITE® CT10, LX20, LX 207, LXA8, LXA10, LXA17, LXA680, LXA1600, LXA1 180, LXA81, LXA816, LXA817, LXA8302, LXA88, LXS868, and AB-8. In another aspect, the hyper-crosslinked polymer is PUROSORB® PAD600 (polydivinylbenzene macroporous, adsorbent resin, non-ionic form).

[0250] In other aspects, the hyper-crosslinked polymer is a carbonaceous material, polystyrene, polyacrylic, polyacrylic ester, cation exchange resin, or polystyrene-divinylbenzene. Non-limiting examples include Amberlite (Rohm and Haas) XAD-2, XAD-4, XAD-7, XAD-16, XAD- 18, XAD- 1180, XAD- 1600, XAD-2000, XAD-2010; Amberchrom (Toso Haas) CG-71m, CG-71c, CG-161m, CG161c; Diaion Sepabeads (Mitsubishi Chemicals) HP20, SP206, SP207, SP850, HP2MG, HP20SS, SP20MS; Dowex (Dow Chemical) XUS-40285, XUS-40323, XUS-43493 (also referred to as Optipore V493 (dry form) or Optipore L493 (hydrated form)), Optipore V503, Optipore SD-2; Hypersol Macronet (Purolite) MN-100, MN-102, MN-150, MN-152, MN-170, MN-200, MN-202, MN-250, MN-252, MN-270, MN-300, MN-400, MN-500, MN- 502, Purosorb (Purolite) PAD 350, PAD 400, PAD 428, PAD 500, PAD 550, PAD 600, PAD 700, PAD 900, and PAD 950. pH Buffering Agent pH buffering agents are substances that may be added to the reaction solution to adjust and / or maintain the pH value. pH buffering agents may be one or more acids, bases, and / or salts. Exemplary and non-limiting pH buffering agents include formic acid, acetic acid, propanoic acid, carbonic acid, fumaric acid, benzoic acid, oxalic acid, malonic acid, maleic acid, succinic acid, lactic acid, gluconic acid, adipic acid, o-toluic acid, benzene tetracarboxylic acid, citric acid, isocitric acid, aconitic acid, propane- 1,2, 3 -tricarboxylic acid, itaconic acid, citraconic acid, hydrogen chloride (HC1), sulfuric acid (H2SO4), nitric acid (HNO3), phosphoric acid (H3PO4), sodium hydroxide (NaOH), potassium hydroxide (KOH), monopotassium phosphate (KH2PO4), dipotassium phosphate (K2HPO4), A-cyclohexyl-2-aminoethanesulfonic acid (CHES), borate, sulfamic acid (H3NSO3), monosodium phosphate (NaH2PO4), disodium phosphate (Na2HPO4), sodium chloride (NaCl), potassium chloride (KC1), calcium chloride (CaCh), magnesium chloride (MgCb), ammonia, [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), A,A-Bis(2- hydroxyethyl)glycine (Bicine), tris(hydroxymethyl)aminomethane (Tris), A-[ l ,3-Dihydroxy-2- (hydroxymethyl)propan-2-yl]glycine (Tricine), 3-[[l,3-dihydroxy-2-(hydroxymethyl)propan-2- yl] amino] -2-hydroxypropane-l -sulfonic acid (TAPSO), 2-[4-(2-Hydroxyethyl)piperazin-l- yl] ethane- 1 -sulfonic acid (HEPES), 2-{[l,3-Dihydroxy-2-(hydroxymethyl)propan-2- yl]amino} ethane- 1 -sulfonic acid (TES), 3-(A-morpholino)propanesulfonic acid (MOPS), piperazine- / / , M -bis(2-ethanesulfonic acid) (PIPES), and 2-(A-morpholino)ethanesulfonic acid (MES).

[0251] By adding one or more pH buffering agents to the reaction solution, the pH value of the reaction solution may be optimized. Typically, the reaction solution may have a pH value of between about 4.0 to about 10.0, between about 4.0 to about 9.5, between about 4.0 to about 9.0, between about 4.0 to about 8.5, between about 4.0 to about 8.0, between about 4.0 to about 7.5, between about 4.0 to about 7.0, between about 4.0 to about 6.75, between about 4.0 to about 6.5, between about 4.25 to about 6.5, between about 4.5 to about 6.5, between about 4.75 to about 6.5, between about 5.0 to about 6.5, between about 5.0 to about 6.25, or between about 5.0 to about 6.0.

[0252] Halide Salt

[0253] As previously described, VHPO enzymes catalyze the formation of organo-halogens in the presence of H2O2 and halide anions (fluoride, iodide, chloride and bromide). Therefore, the halogenation of the organic substrates may be further controlled by the addition of one or more halide anion. The one or more halide anion may be added in the form of one or more halide salt. Exemplary and non-limiting halide salts include potassium chloride (KC1), potassium bromide (KBr), potassium iodide (KI), sodium chloride (NaCl), sodium bromide (NaBr), and sodium iodide (Nal).

[0254] Alkaline Earth Metal Salt

[0255] The halogenation reaction may also be controlled by addition of one or more alkaline earth metal ions. For example, the addition of Ca2+and / or Mg2+may induce precipitation of certain byproducts such as dimethylglutaric acid, thereby reducing the amount of unwanted by-products in the reaction solution. The one or more alkaline earth metal ions may be added in the form of one or more alkaline earth metal salt. Exemplary and non-limiting alkaline earth metal salts include calcium oxide (CaO), calcium acetate, calcium carbonate (CaCCb), calcium chloride (CaCh), calcium citrate, calcium gluconate, calcium sulfate (CaSO-i), calcium bromide (CaBn), calcium hydroxide (Ca(OH)2), calcium nitrate (Ca(NOs)2), magnesium oxide (MgO), magnesium acetate, magnesium carbonate (MgCCh), magnesium chloride (MgCb), magnesium citrate, magnesium gluconate, magnesium sulfate (MgSC ), magnesium bromide (MgBn), magnesium hydroxide (Mg(0H)2), and magnesium nitrate (Mg(NOs)2).

[0256] Ultrafiltration Membrane

[0257] To remove the unwanted by-products in the reaction solution, an ultrafiltration cassette may be installed to the device. The ultrafiltration cassette may include an ultrafiltration membrane to filtrate the reaction solution and remove the unwanted by-products. The ultrafiltration membrane may be made of various materials known in the field that are suitable for use in filtration. Typically, the ultrafiltration membrane is made of one or more polymer materials. Exemplary and non-limiting materials suitable for making the ultrafiltration membrane include cellulose, hemicellulose, cellulose acetate, regenerated cellulose, polyether sulfone (PES), polysulfone (PSU), polyphenylene sulfone (PPSU), poly(bisphenol-A sulfone) (PSF), poly(arylene sulfone) (PAS), polylactide, poly(aryl ether ether ketone) (PEEK), polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET), polyethylene chlorotrifluoroethylene (PECTFE), polyethylene tetrafluoroethylene (PETFE), polytetrafluoroethylene (PTFE), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), polyvinylidene fluoride (PVDF), thermoplastic polyurethane (TPU), polyvinyl-fluoride, ethylenechlorotrifluoroethylene copolymer, ethylene-tetrafluoroethylene-copolymer, fluoroethylenepropylene copolymer, perfluoralkoxy polymer, and / or polychloro-trifluoroethylene.

[0258] Desiccant or Water Absorbing Material

[0259] The reaction solution typically contains water. When the air flow passes through the reaction solution, a trace amount of moisture / water vapor may be brought together with the trihalomethane products. Thus, the trace amount of moisture / water vapor may be captured by the tank / high surface area materials along with the trihalomethane products. This may reduce the quality of the captured trihalomethane products as water may contribute to the degradation of trihalomethanes.

[0260] To solve this issue, one or more desiccant or water absorbing material may be used to remove the moisture / water vapor contained in the air flow before capturing the trihalomethane products with high surface area materials. Exemplary and non-limiting materials suitable for use as desiccant or water absorbing material include activated alumina, calcium chloride (CaCh), calcium oxide (CaO), calcium hydride (CaFF), calcium sulfate (CaSO-i), cobalt(II) chloride (C0CI2), copper(II) sulfate (CuSCh), lithium chloride (LiCl), lithium bromide (LiBr), magnesium chloride (MgCh), magnesium sulfate (MgSC ), magnesium perchlorate (Mg(C104)2), Nafion, phosphorus pentoxide (P4O10), potassium carbonate (K2CO3), potassium hydroxide (KOH), sodium hydroxide (NaOH), silica gel, sodium, sodium chlorate (NaQOs), sodium chloride (NaCl), sodium sulfate (Na2SO4), sucrose, sulfuric acid (H2SO4), and triethylene glycol. Animal Feed

[0261] Also disclosed herein is an animal feed comprising the trihalomethanes produced by the invention described herein. The animal feed may be solid (e.g., powder, granules, pellets, feed block, lick block), semi-solid (e.g., gel, ointment, cream, paste) or liquid (e.g., solutions, suspensions, emulsions). The animal feed may independently be solid, semi-solid (e.g., gel, ointment, cream, paste) or liquid (e.g., solutions, suspensions, emulsions). For example, the animal feed may both be liquid or both be semi-solid or both be solid. Alternatively, the animal feed and composition may each be a different physical state. For example, the animal feed may be solid or semi-solid and the composition may be liquid. The composition may, for example, be used to “top- dress” (added on top) a ruminant feedlot ration or may be used to blend into a total mixed ration. In one aspect, the animal feed is in the form of a feed block. In another aspect, the animal feed is in the form of a lick block. In another aspect, the animal feed is in the form of a low moisture block.

[0262] The composition may, for example, be added to the drinking water of the animal. In certain embodiments, the composition may be added to the drinking water of the animal immediately before ingestion, for example up to 1 hour before ingestion or up to 30 minutes before ingestion or up to 15 minutes before ingestion or up to 5 minutes before ingestion.

[0263] The three main types of animal feed include roughages, concentrates and mixed feeds. In general, roughages contain a higher percentage of crude fiber and a lower percentage of digestible nutrients than concentrates. For example, roughages may be defined as containing equal to or greater than 20 wt% crude fiber and equal to or less than 60 wt% total digestible nutrients. Roughages may include, for example, dry roughages (e.g., hay, straw, artificially dehydrated forages containing at least 90 wt% dry matter), silages (formed from green forages such as grass, alfalfa, sorghum and corn and preserved in a silo at dry matter contents of 20 to 50 %), and pastures (e.g., green growing pastures providing forage that has a high water content and generally less than 30 % dry matter). The two basic types of roughages include grasses and legumes. Grasses are generally higher in fiber and dry matter than legumes. Legumes are generally higher in proteins, metabolizable energy, vitamins and minerals. Concentrates contain a relatively lower percentage of crude fiber and a higher percentage of digestible nutrients than roughages. For example, concentrates may be defined as containing less than 20 wt% crude fiber and greater than 60 wt% total digestible nutrients. Concentrates may include, for example, energy-rich grains and molasses. Corn, wheat, oats, barley and milo (sorghum grain) are energy-rich grains, containing about 70 to 80 wt% total digestible nutrients.

[0264] Mixed feeds are generally a mixture of roughages and concentrates to provide "complete" balanced rations and may be either high or low in energy, protein or fiber. The disclosed compositions (e.g., the one or more high surface area material with trihalomethanes captured therein), for example, can be combined with animal feed in various amounts depending on the total amount of fungal strain, culture supernatant from the fungal strain, or combination thereof intended to be administered to the animal.

[0265] The animal feed may, for example, comprise from about 0.0001 wt% to about 10 wt% of disclosed compositions, based on the total dry weight of the animal feed. The animal feed may, for example, comprise from about 0.01 wt% to about 10 wt% of disclosed composition, based on the total dry weight of the animal feed. For example, the animal feed may comprise from about 0.001 wt% to about 9.5 wt%, or from about 0.005 wt% to about 9 wt%, or from about 0.01 wt% to about 8.5 wt%, or from about 0.05 wt% to about 8 wt%, or from about 0.1 wt% to about 7.5 wt%, or from about 0.9 wt% to about 7 wt%, or from about 1 wt% to about 6 wt%, or from about

[0266] 1.5 wt% to about 5.5 wt%, or from about 2 wt% to about 5 wt%, or from about 2.5 wt% to about

[0267] 4.5 wt%, or from about 3 wt% to about 4 wt% disclosed composition based on the total dry weight of the animal feed. For example, the animal feed may comprise from about 0.4 wt% to about 9.5 wt%, or from about 0.5 wt% to about 9 wt%, or from about 0.6 wt% to about 8.5 wt%, or from about 0.7 wt% to about 8 wt%, or from about 0.8 wt% to about 7.5 wt%, or from about 0.9 wt% to about 7 wt%, or from about 1 wt% to about 6 wt%, or from about 1.5 wt% to about 5.5 wt%, or from about 2 wt% to about 5 wt%, or from about 2.5 wt% to about 4.5 wt%, or from about 3 wt% to about 4 wt% disclosed composition based on the total dry weight of the animal feed.

[0268] In one embodiment, the disclosed composition is administered at a dose of preferably at least 16.67, 10, 5, 3, 2, 1, 0.5, 0.25 0.125 or 0.067% of the dry matter administered to the ruminant animal. For example, if a 450 kg ruminant animal (e.g., steer) consumes 2.5% to 3% of its body weight per day of feed, then the disclosed composition is administered at a dose proportional to the amount of dry matter administered to the ruminant. In the case of a 450 kg ruminant animal, and where 80% of the feed is dry matter, if the animal consumes about 2.5% of its body weight per day, then the disclosed composition is administered at a dose of about 0.27, 0.18, 0.09, 0.045, 0.0225, 0.01125 or 0.00603 kg per day to result in a dose at least 3, 2, 1, 0.5, 0.25 0.125 or 0.067wt% of the dry matter administered to the ruminant animal.

[0269] In another embodiment, the disclosed composition is administered at a dose that provides 1 to 100 mg bromoform per kg dry matter intake, 1 to 90 mg bromoform per kg dry matter intake, 1 to 80 mg bromoform per kg dry matter intake, 1 to 70 mg bromoform per kg dry matter intake, 1 to 60 mg bromoform per kg dry matter intake, 1 to 50 mg bromoform per kg dry matter intake, 10 to 100 mg bromoform per kg dry matter intake, 10 to 90 mg bromoform per kg dry matter intake, 10 to 80 mg bromoform per kg dry matter intake, 10 to 70 mg bromoform per kg dry matter intake, 10 to 60 mg bromoform per kg dry matter intake, 10 to 50 mg bromoform per kg dry matter intake, 25 to 100 mg bromoform per kg dry matter intake, 25 to 90 mg bromoform per kg dry matter intake, 25 to 80 mg bromoform per kg dry matter intake, 25 to 70 mg bromoform per kg dry matter intake, 25 to 60 mg bromoform per kg dry matter intake, or 25 to 50 mg bromoform per kg dry matter intake.

[0270] Any embodiment of the invention is meant to be illustrative only and is not meant to be limiting to the invention. Therefore, it should be appreciated that various other changes and modifications can be made to any embodiment described without departing from the spirit and scope of the invention.

[0271] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0272] The present invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference in their entirety for all purposes.

[0273] EXAMPLES

[0274] Example 1.

[0275] Fig. 1 depicts an exemplary embodiment of the present invention. The exemplary device for producing trihalomethane, as illustrated in Fig. 1, includes a reactor 100 containing a solution 102; a paddle 104 for facilitating the mixing of the solution 102 in the reactor 100; a first reservoir 10 connecting to the reactor 100 via a first channel 11; a second reservoir 20 connecting to the reactor 100 via a second channel 21; a fourth reservoir 40 connecting to the reactor 100 via a fourth channel 41; an air pump 60 connecting to the reactor 100 via a gas inflow channel 61; an ultrafiltration cassette 80 connecting to the reactor 100 via a liquid outflow channel 81 and a liquid inflow channel 83; and a tank 70 connecting to the reactor 100 via a gas outflow channel 71.

[0276] The reactor 100 contains the solution 102. The reactants (e.g., ketones and diketones) and the at least one VHPO enzyme are added to the solution 102 in the reactor. Within the reactor 100, the at least one VHPO enzyme catalyzes the formation of organo-halogens in the presence of H2O2 and halide anions (fluoride, iodide, chloride and bromide). The reactor 100 can be any shape that can contain a solution. In some embodiments, the reactor 100 has a cylindrical shape. In some other embodiments, the reactor 100 has a spherical shape. In yet some other embodiments, the reactor 100 has a rectangular shape.

[0277] The reactor 100 can be any size that is suitable for carrying out the halogenation reaction. For example, the reactor 100 can have a volume of about 1 m3, about 5 m3, about 10 m3, about 15 m3, about 20 m3, about 25 m3, about 50 m3, about 75 m3, about 100 m3, about 125 m3, about 150 m3, about 175 m3, about 200 m3, about 225 m3, about 250 m3, about 275 m3, about 300 m3, about 325 m3, about 350 m3, about 375 m3, about 400 m3, about 425 m3, about 450 m3, about 475 m3, about 500 m3, about 525 m3, about 550 m3, about 575 m3, about 600 m3, about 625 m3, about 650 m3, about 675 m3, about 700 m3, about 725 m3, about 750 m3, about 775 m3, about 800 m3, about 825 m3, about 850 m3, about 875 m3, about 900 m3, about 925 m3, about 950 m3, about 975 m3, or about 1000 m3.

[0278] The at least one VHPO enzyme is disposed within the reactor 100. To prevent the at least one VHPO enzyme from escaping the reactor 100 and / or entering other places, the at least one VHPO enzyme is immobilized and / or confined in the reactor 100. Techniques for immobilizing and / or confining the at least one VHPO enzyme that are suitable to be applied in the present invention are described above.

[0279] To facilitate the halogenation reaction, the reactor 100 may comprise one or more mixers. For example, a paddle 104 can be installed to the reactor 100. The mixing / blending of the reactants and the at least one VHPO enzyme in the solution 102 can be facilitated by rotating the paddle 104. In some other embodiments, the reactor 100 can comprise an agitator blender, a dual shaft blender, a ribbon blender, a rotary blender, a vibrational blender, a static mixer, and / or a vertical blender. The first reservoir 10 is connected to the reactor 100 via a first channel 11 and contains one or more organic substrates that may be halogenated by the at least one VHPO enzyme. The one or more organic substrates may be one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV:

[0280] F ormula III F ormula IV

[0281] . In some embodiments, the one or more organic substrates may be a compound of Formula II. In some embodiments, in Formula II, R3, R4, Rs, Rs, R7, and Rs each independently represents hydrogen, halogen, hydroxyl, carboxyl, substituted or unsubstituted Ci-Ce alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or combinations thereof, wherein one or more methylene groups of the substituted or unsubstituted Ci-Ce alkyl of R3, R4, Rs, Rs, R7, and / or Rs may optionally be replaced by -O-, -NH-, -S-, -COO-, -COS-, CONH-, -CSNH-, and / or -CSO-. In some embodiments, the compound of Formula II is one or more of the following:

[0282]

[0283] . In some embodiments, the compound of Formula II is Formula Il-d.

[0284] The first channel 11 fluidly connects the first reservoir 10 with the reactor 100. During operation of the device for producing trihalomethane, the first reservoir 10 may transfer the organic substrates (e.g., one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV) to the reactor 100 through the first channel 11. Depending on the progress of the reaction (halogenation), the organic substrates may be transferred to the reactor intermittently or continuously. In some embodiments, a rate of transfer of the organic substrates from the first reservoir 10 to the reactor 100 may be about 1 g / min, about 2.5 g / min, about 5 g / min, about 7.5 g / min, about 10 g / min, about 12.5 g / min, about 15 g / min, about 17.5 g / min, about 20 g / min, about 22.5 g / min, about 25 g / min, about 27.5 g / min, about 30 g / min, about 32.5 g / min, about 35 g / min, about 37.5 g / min, about 40 g / min, about 42.5 g / min, about 45 g / min, about 47.5 g / min, about 50 g / min, about 55 g / min, about 60 g / min, about 65 g / min, about 70 g / min, about 75 g / min, about 80 g / min, about 85 g / min, about 90 g / min, about 95 g / min, about 100 g / min, about 110 g / min, about 120 g / min, about 130 g / min, about 140 g / min, about 150 g / min, about 160 g / min, about 170 g / min, about 180 g / min, about 190 g / min, about 200 g / min, about 300 g / min, about 400 g / min, about 500 g / min, about 600 g / min, about 700 g / min, about 800 g / min, about 900 g / min, about 1000 g / min, about 2 kg / min, about 3 kg / min, about 4 kg / min, about 5 kg / min, about 6 kg / min, about 7 kg / min, about 8 kg / min, about 9 kg / min, or about 10 kg / min.

[0285] As previously described, VHPO enzymes catalyze the formation of organo-halogens in the presence of H2O2 and halide anions (fluoride, iodide, chloride and bromide). Therefore, the halogenation of the organic substrates may be further controlled by the addition of one or more halide anion. The one or more halide anion may be added to the solution 102 / reactor 100 separately or together with the addition of the organic substrates. In some embodiments, the one or more halide anion is added in the form of one or more halide salt. For example, the one or more halide salt may be selected from the group consisting of: potassium chloride (KC1), potassium bromide (KBr), potassium iodide (KI), sodium chloride (NaCl), sodium bromide (NaBr), and sodium iodide (Nal). In some embodiments, the one or more halide anion is added together with the addition of the organic substrates. For example, the addition of the one or more halide anion may be accomplished by adding the one or more halide salt from the first reservoir 10 to the solution 102 / reactor 100.

[0286] The second reservoir 20 is connected to the reactor 100 via a second channel 21 and contains hydrogen peroxide. The second channel 21 fluidly connects the second reservoir 20 with the reactor 100. During operation of the device for producing trihalomethane, the second reservoir 20 may transfer the hydrogen peroxide to the reactor 100 through the second channel 21. Depending on the progress of the reaction (halogenation), the hydrogen peroxide may be transferred to the reactor intermittently or continuously. In some embodiments, a rate of transfer of the hydrogen peroxide from the second reservoir 20 to the reactor 100 may be about 1 g / min, about 2.5 g / min, about 5 g / min, about 7.5 g / min, about 10 g / min, about 12.5 g / min, about 15 g / min, about 17.5 g / min, about 20 g / min, about 22.5 g / min, about 25 g / min, about 27.5 g / min, about 30 g / min, about 32.5 g / min, about 35 g / min, about 37.5 g / min, about 40 g / min, about 42.5 g / min, about 45 g / min, about 47.5 g / min, about 50 g / min, about 55 g / min, about 60 g / min, about 65 g / min, about 70 g / min, about 75 g / min, about 80 g / min, about 85 g / min, about 90 g / min, about 95 g / min, about 100 g / min, about 110 g / min, about 120 g / min, about 130 g / min, about 140 g / min, about 150 g / min, about 160 g / min, about 170 g / min, about 180 g / min, about 190 g / min, about 200 g / min, about 300 g / min, about 400 g / min, about 500 g / min, about 600 g / min, about 700 g / min, about 800 g / min, about 900 g / min, about 1000 g / min, about 2 kg / min, about 3 kg / min, about 4 kg / min, about 5 kg / min, about 6 kg / min, about 7 kg / min, about 8 kg / min, about 9 kg / min, or about 10 kg / min.

[0287] The fourth reservoir 40 is connected to the reactor 100 via a fourth channel 41 and contains at least one pH buffering agent. The fourth channel 41 fluidly connects the fourth reservoir 40 with the reactor 100. During operation of the device for producing trihalomethane, the fourth reservoir 40 may transfer the at least one pH buffering agent to the reactor 100 through the fourth channel 41. Depending on the progress of the reaction (halogenation), the at least one pH buffering agent may be transferred to the reactor intermittently or continuously. In some embodiments, a rate of transfer of the at least one pH buffering agent from the fourth reservoir 40 to the reactor 100 may be about 1 g / min, about 2.5 g / min, about 5 g / min, about 7.5 g / min, about 10 g / min, about 12.5 g / min, about 15 g / min, about 17.5 g / min, about 20 g / min, about 22.5 g / min, about 25 g / min, about 27.5 g / min, about 30 g / min, about 32.5 g / min, about 35 g / min, about 37.5 g / min, about 40 g / min, about 42.5 g / min, about 45 g / min, about 47.5 g / min, about 50 g / min, about 55 g / min, about 60 g / min, about 65 g / min, about 70 g / min, about 75 g / min, about 80 g / min, about 85 g / min, about 90 g / min, about 95 g / min, about 100 g / min, about 110 g / min, about 120 g / min, about 130 g / min, about 140 g / min, about 150 g / min, about 160 g / min, about 170 g / min, about 180 g / min, about 190 g / min, about 200 g / min, about 300 g / min, about 400 g / min, about 500 g / min, about 600 g / min, about 700 g / min, about 800 g / min, about 900 g / min, about 1000 g / min, about 2 kg / min, about 3 kg / min, about 4 kg / min, about 5 kg / min, about 6 kg / min, about 7 kg / min, about 8 kg / min, about 9 kg / min, or about 10 kg / min. An ultrafiltration cassette 80 may be connected with the reactor 100 to allow for removing by-products of the reaction (halogenation). The by-products of the reaction (halogenation) may be removed by filtering the solution 102 via the ultrafiltration cassette 80. The ultrafiltration cassette 80 is connected to the reactor 100 via a liquid outflow channel 81 and a liquid inflow channel 83. The solution 102 flows from the reactor 100 to the ultrafiltration cassette 80 through the liquid outflow channel 81 such that the solution 102 is filtered by the ultrafiltration cassette 80; and then the solution 102 flows from the ultrafiltration cassette 80 to the reactor 100 through the liquid inflow channel 83.

[0288] Within the ultrafiltration cassette 80, an ultrafiltration membrane (not showing on the drawings) may be installed to facilitate the filtration of the solution 102. In addition to filtering the solution 102, the ultrafiltration membrane may also function as a confinement to the at least one VHPO enzyme. In other words, with the installation of the ultrafiltration membrane, the at least one VHPO enzyme may be retained in the solution 102 / reactor 100 during the filtration of the solution 102.

[0289] An air pump 60 may be connected to the reactor 100 to pump air into the reactor 100. By pumping air into the reactor 100, an air flow may be created, which may carry away with the air flow the trihalomethanes produced by the reaction (halogenation). The gas inflow channel 61 connects the air pump 60 with the reactor 100. The gas inflow channel 61 may be connected to the reactor 100 at any position. For example, in some embodiments, the gas inflow channel 61 may be connected to the reactor 100 at a bottom position of the reactor 100 and be submerged under the solution 102. In some other embodiments, the gas inflow channel 61 may be connected to the reactor 100 at an upper position and not being submerged under the solution 102.

[0290] The trihalomethanes produced by the reaction (halogenation) are separated from the solution 102 and be carried out from the solution 102 / reactor 100 by the air flow. In some embodiments, a tank 70 can be connected to the reactor 100 for capturing the trihalomethanes. For example, in some embodiments, a gas outflow channel 71 connects the tank 70 with the reactor 100. when the device is in operation, an air flow is pumped into the solution 102 / reactor 100 by the air pump 60; the trihalomethanes produced by the reaction (halogenation) are then separated from the solution 102 and be carried away with the air flow; and the air flow is directed to the tank 70 by passing through the gas outflow channel 71. To facilitate capturing of the trihalomethanes, the tank 70 may contain one or more high surface area material, such that the trihalomethanes are absorbed / captured by the one or more high surface area material. In addition, the tank 70 may be specifically designed to facilitate capturing of the trihalomethanes. For example, the tank 70 may be a rotating drum, such that during operation of the device, the tank 70 may rotate and thus provide further mixing of the air flow / trihalomethanes with the one or more high surface area material.

[0291] FIG. 2 depicts another exemplary embodiment of the present invention. The exemplary device for producing trihalomethane, as illustrated in FIG. 2, further includes a third reservoir 30 to receive the one or more organic substrates transferred from the first reservoir 10 via the first channel 11 , the hydrogen peroxide transferred from the second reservoir 20 via the second channel 21, and / or the at least one pH buffering agent transferred from the fourth reservoir 40 via the fourth channel 41. The substances received from the first reservoir 10, the second reservoir 20, and / or the fourth reservoir 40 are mixed in the third reservoir 30 to form a mixture, which is subsequently transferred to the reactor 100 via a third channel 31. This alternative design ensures that the reactants are fully mixed, and the pH value controlled, before the halogenation reaction being catalyzed by the at least one VHPO enzyme. By doing so, the halogenation reaction can be carried out in a more smoothly manner.

[0292] FIG. 3 depicts yet another exemplary embodiment of the present invention. The exemplary device for producing trihalomethane, as illustrated in FIG. 3, further includes a fifth reservoir 50. The reactor 100 is connected to the fifth reservoir 50 via a gas outflow channel 51, which in turn is connected to the tank 70 via a gas outflow channel 71. The fifth reservoir 50 may contain at least one desiccant or water absorbing material (e.g., activated alumina, Nafion, silica gel) so as to remove a moisture of the air flow when the air flow is passing from the reactor 100 to the tank 70. By doing so, the moisture / water contained in the air flow may be removed. This reduces or removes the amount of water captured by the one or more high surface area material contained in the tank 70, thereby resulting in lesser degradation of trihalomethanes.

[0293] FIG. 4 depicts a further exemplary embodiment of the present invention. The exemplary device for producing trihalomethane, as illustrated in FIG. 4, further includes a first pump 12, a second pump 22, a third pump 32, a fourth pump 42, and / or a filtration pump 82. The first pump 12 facilitates the transfer of the one or more organic substrates and / or the one or more halide salt from the first reservoir 10 to the third reservoir 30 and / or the reactor 100; the second pump 22 facilitates the transfer of the hydrogen peroxide from the second reservoir 20 to the third reservoir 30 and / or the reactor 100; the fourth pump 42 facilitates the transfer of the at least one pH buffering agent to the third reservoir 30 and / or the reactor 100; and the third pump 32 facilitates the transfer of the mixture from the third reservoir 30 to the reactor 100. The filtration pump 82 facilitates transfer of the solution 102 from the reactor 100 to the ultrafiltration cassette 80 and / or the transfer of the solution 102 from the ultrafiltration cassette 80 to the reactor 100.

[0294] The halogenation reaction may also be controlled by addition of one or more alkaline earth metal ions. For example, alkaline earth metal ions such as Ca2+and / or Mg2+may be added to the solution 102. The addition of Ca2+and / or Mg2+may induce precipitation of certain by-products such as dimethylglutaric acid, thereby reducing the amount of unwanted by-products in the solution 102. The one or more alkaline earth metal ions may be added to the solution 102 in the form of one or more alkaline earth metal salt. The one or more alkaline earth metal salt may comprise: calcium oxide (CaO), calcium acetate, calcium carbonate (CaCO,), calcium chloride (CaCh), calcium citrate, calcium gluconate, calcium sulfate (CaSC ), calcium bromide (CaBn), calcium hydroxide (Ca(OH)2), calcium nitrate (Ca(NOs)2), magnesium oxide (MgO), magnesium acetate, magnesium carbonate (MgCCh), magnesium chloride (MgCb), magnesium citrate, magnesium gluconate, magnesium sulfate (MgSC ), magnesium bromide (MgBn), magnesium hydroxide (Mg(0H)2), magnesium nitrate (Mg(NOs)2), and / or a combination thereof.

[0295] FIG. 5 depicts an alternative exemplary embodiment of the present invention. The exemplary device for producing trihalomethane, as illustrated in FIG. 5, further includes a sixth reservoir 15 (reaction controlling reservoir 15). The sixth reservoir 15 is connected to the reactor 100 and contains the one or more alkaline earth metal salt. During operation of the device, the sixth reservoir 15 may transfer the one or more alkaline earth metal salt to the reactor 100, such that the one or more alkaline earth metal salt is dissolved in the solution 102. The sixth reservoir 15 may also contain other substances for controlling the halogenation reaction. For example, in some alternative embodiments, the sixth reservoir 15 may contain the one or more halide salt, such that additional halide anion(s) may be transferred to the solution 102 / reactor 100 when needed.

[0296] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth. Example 2.

[0297] In a 20 L plastic container, a 0.1 M HEPES buffer was made by mixing 477 g of HEPES with 20 L of distilled water. Using a pH meter, the pH of the buffer was recorded and adjusted to 6.5, using either acetic acid or sodium hydroxide solution. The same buffer will be used to prepare the hydrogen peroxide / potassium bromide and dimedone reagent.

[0298] Reagent preparation:

[0299] Dimedone has a water solubility of approximately 4 g / L. Therefore, a 15 L solution of dimedone was prepared by dissolving 60 g of dimedone (or 0.43 moles) in 15 L of 0.1 M HEPES buffer pH 6.5. After ensuring complete mixing of the substance, the pH of the solution was tested again and adjusted to 6.5, if necessary. The remaining 5 L of the buffer solution was used to make the potassium bromide and hydrogen peroxide solution together. Approximately around 152.8 g of KBr (1.29 moles, 3 equivalents relative to dimedone) and 150 mL of 35% H2O2 (1.75 moles, 4 equivalents relative to dimedone) is added into the 5 L buffer and shaken to mix the sample. For continuous operation, new batches of reagent solution were freshly prepared. A 3: 1 stoichiometric ratio of potassium bromide to dimedone is maintained to minimize VHPO enzyme inhibition resulting from an elevated potassium bromide concentration. As the reagent is consumed, new batches of reagent solution were prepared immediately prior to their use.

[0300] VHPO enzyme was manufactured through a fungal fermentation pathway and filtered to remove biomass. Approximately, 3 L of the enzyme was transferred into a large 5 L Erlenmeyer flask and placed on a magnetic stirrer. With stirring, 71.5 g of HEPES (resulting in a 0.1 M buffer strength) was added into the enzyme supernatant and the pH adjusted accordingly to 6.5. Following this, 120 mL 0.025 M of sodium vanadate (ImM final concentration for a 3L enzyme) solution was later added into the enzyme supernatant to activate the enzyme. The enzyme was transferred into the reactor.

[0301] A tangential flow filtration (regenerated cellulose, ultrafiltration with a 3 kDa molecular weight cutoff, 0.11 m2area) was integrated into the reactor setup. The 3 kDa molecular weight cutoff was selected to permit the removal of excess water and byproducts from inside the reactor while retaining the VHPO enzyme (20 kDa) within the reactor. The filtration process was driven by a peristaltic pump. The peristaltic pump rpm and the pressure regulator was maintained as such to regulate the pressure within the filtration system around 0.1 -0.2 MPa. The reactor is a cylinder with 7 airtight Swagelok tube fittings. Two of the fittings have tubes that deliver the reagents (dimedone and KBr / EfcCh) dropwise into the reactor via two peristaltic pumps. Another fitting is connected to an air pump with its tube reaching the bottom of the reactor; air is vigorously pump into the reactor to mix the reagents and enzyme thoroughly. Furthermore, the continuous purging of air also volatilizes the bromoform in the supernatant where it is directed via another tube fitting into a separate container holding the activated carbon carrier. The container holding the activated carbon is placed on top of a ball mill / roller, which can rotate the activated carbon container allowing for a homogeneous deposition of bromoform. The rotation of the container is required for a uniform gas phase deposition of bromoform throughout the activated carbon. The next two fittings are connected to the feed inlet of the ultrafiltration membrane (with a tube to the bottom of the reactor) and the retentate line of the ultrafiltration membrane. The last connector is fitted to a peristaltic pump where acetic acid can be added via a peristaltic pump. A pH probe can be utilized to control the addition of acid into the reactor to maintain constant pH conditions. After reaching a 15 L liquid volume in the 20 L reactor, the permeate flow rate of the ultrafiltration membrane was tuned such that the reactor volume maintained a constant liquid volume. This was obtained by using a feedback loop between a weight scale (which measures the weight of the reactor) and a peristaltic pump for the ultrafiltration membrane setup (which controls the permeate flow rate). In this example, the reagents were added over a period of 48 hours. Two new batches of reagents were subsequently added over a period of 96 hours. Using a chlorobenzene extract method and subsequent GCMS analysis of this extract, we showed that a 12.4 wt.% loading of bromoform onto activated carbon could be obtained.

[0302] Example 3.

[0303] Activated carbon will also absorb a significant amount of water vapor. For quantification and feed stability reasons, it is desirable to have a bromoform loaded material that has a low moisture content. As a significant of water vapor is entrained along with the bromoform as it is carried into the activated carbon deposition chamber, a way to selectively remove the moisture prior to reaching the deposition is necessary.

[0304] FIG. 6 depicts an exemplary device for producing trihalomethane according to the present invention. The exemplary device illustrated in FIG. 6 demonstrates how the water vapor may be removed before capturing the bromoform. A first reservoir 10 is connected to the reactor 100 via a first channel 11. A pump is installed within the first reservoir 10, facilitating it to transfer dimedone and KBr to the reactor 100, such that sufficient substrates and reactants are provided for the reaction. A second reservoir 20 is connected to the reactor 100 via a second channel 21. A pump is installed within the second reservoir 20, facilitating it to transfer hydrogen peroxide to the reactor 100, such that sufficient reactants are provided for the reaction. A fourth reservoir 40 is connected to the reactor 100 via a fourth channel 41. A pump is installed within the fourth reservoir 40, facilitating it to transfer acetic acid to the reactor 100, such that the pH condition of the solution 102 is properly maintained. An ultrafiltration cassette 80 is connected with the reactor 100 to allow for removing by-products of the reaction (halogenation). The ultrafiltration cassette 80 is connected to the reactor 100 via a liquid outflow channel 81 and a liquid inflow channel 83. A filtration pump is installed to the liquid outflow channel 81, facilitating the solution 102 to flow from the reactor 100 to the ultrafiltration cassette 80 through the liquid outflow channel 81, and then flowing from the ultrafiltration cassette 80 to the reactor 100 through the liquid inflow channel 83.

[0305] An air pump 60 is connected to the reactor 100 to pump air into the reactor 100 via a gas inflow channel 61. By pumping air into the reactor 100, an air flow may be created, which may carry away with the air flow the trihalomethanes (bromoform) produced by the reaction (halogenation). A gas outflow channel 71 connects the tank 70 with the reactor 100. When the device is in operation, an air flow is pumped into the reactor 100 by the air pump 60; the bromoform produced by the reaction (halogenation) are then separated from the solution 102 and be carried away with the air flow; and the air flow is directed to the tank 70 by passing through the gas outflow channel 71. The tank 70 is a rotating drum and contains activated charcoal / activated carbon for the bromoform.

[0306] As previously discussed, the air flow carrying the bromoform may also contain water vapor, which can also be captured by activated charcoal / activated carbon. To remove the water vapor, a Nafion drying tube 50 (fifth reservoir 50) is installed to the gas outflow channel 71 in a manner depicted in FIG. 6. In particular, an outlet is installed to connect the tank 70 and the Nafion drying tube 50. This outlet may direct the dry air flow to flow from the tank 70 to the housing of the Nafion drying tube 50, facilitating the dehydration process. The air flow containing water vapor will then be discharged from the Nafion drying tube 50. By doing so, the water vapor will be removed from the air flow midway within the gas outflow channel 71, and the amount of water vapor being captured by activated charcoal / activated carbon may be greatly reduced.

[0307] Example 4.

[0308] 500 mL of a fungal-derived VHPO enzyme was added to a 50 L reactor containing 0.1 M HEPES buffer. This VHPO enzyme shows good bromination activity between pH values of 5 to 7 with an optimal value around 6. The hydrolysis step in the haloform reaction favors an alkaline environment. Batch experiments have shown that pH values from 6.5 to 7.5 are a good compromise between the requirements for both reactions necessary to produce bromoform. As such, in this example, pH values of 6.8, 6.9, 7.0 and 7.1 were considered. Based on the reaction stoichiometry, it was evident that the reaction would consume acid and as such an acid (in this case, acetic acid) would need to be added as a reagent to maintain constant pH conditions. Based on the reaction stoichiometry, the ratio of reagents required to maintain a constant pH at each value could be calculated using the Henderson-Hasselbach equation for buffers. These calculations assume the pKa value / values of acetic acid, dimedone and 3,3-dimethylglutaric acid are 4.76, 5.23 and 3.85 / 6.45, respectively. Importantly, this calculation assumes that acetic acid, dimedone and the first proton in 3,3-dimethylglutaric acid are fully deprotonated. The second proton in 3,3- dimethylglutaric acid is assumed to have degree of ionization as given in Table 5 below:

[0309] Table 5. Degrees of ionization calculated at various pHs.

[0310] The results of these calculations are given below:

[0311] Stoichiometry of reaction at pH 6.8: 1 deprotonated potassium dimedone (CsHnChK) + 3 KBr + 3 H2O2 + 2.309 acetic acid (CH3CO2H)

[0312] 1 bromoform (CBr,H) + 0.691 dipotassium glutarate (C7H10O4K2) + 0.309 monopotassium glutarate (C7H11O4K) + 2.309 potassium acetate (CH3CO2K) + 4 H2O

[0313] Stoichiometry of reaction at pH 6.9:

[0314] 1 deprotonated potassium dimedone (CsHnChK) + 3 KBr + 3 H2O2 + 2.262 acetic acid (CH3CO2H) 1 bromoform (CBr,H) + 0.738 dipotassium glutarate (C7H10O4K2) + 0.262 monopotassium glutarate (C7H11O4K) + 1.262 potassium acetate (CH3CO2K) + 4 H2O

[0315] Stoichiometry of reaction at pH 7.0:

[0316] 1 deprotonated potassium dimedone (CsHuChK) + 3 KBr + 3 H2O2 + 2.22 acetic acid (CH3CO2H) 1 bromoform (CBr,H) + 0.78 dipotassium glutarate (C7H10O4K2) + 0.22 monopotassium glutarate (C7H11O4K) + 1.22 potassium acetate (CH3CO2K) + 4 H2O

[0317] Stoichiometry of reaction at pH 7.1:

[0318] 1 deprotonated potassium dimedone (CsHi 1O2K) + 3 KBr + 3 H2O2 + 2.183 acetic acid (CH3CO2H) 1 bromoform (CBr,H) + 0.817 dipotassium glutarate (C7H10O4K2) + 0.183 monopotassium glutarate (C7H11O4K) + 1.183 potassium acetate (CH3CO2K) + 4 H2O

[0319] For dimedone, a 50 mM HEPES (MW 238.3, 11.9 g / L) was prepared and adjusted to the desired pH using potassium hydroxide, dimedone at a 4 g / L concentration (28.53 mM) was added to the mixture and homogenized. A low concentration of HEPES buffer was used to minimize costs and to make the system more responsive to pH changes; this latter requirement was necessary as pH control was used to meter the reagents as the reaction progressed. If the buffer concentration was higher this could make the control system less sensitive. The addition of reagent with this control approach was used as a marker of reaction kinetics. For example, if the reaction kinetics decreased over time (e.g., due to loss of enzyme activity), the addition of reagents must also decrease otherwise there would be an excess of acid causing the pH to decrease from the desired value. The other reagents were prepared without the addition of HEPES buffer, and the hydrogen peroxide was stored appropriately until use (i.e., making up more than several days allocation of hydrogen peroxide was avoided). A single peristaltic pump was used with a feedback loop back to a pH meter. This same peristaltic pump simultaneously pumped up to 4 solutions at the same flow rate. Based on the upper solubility of dimedone and the required stoichiometry at each pH value, the concentration of hydrogen peroxide, potassium bromide and acetic acid could be calculated with the results given below in Table 6.

[0320] Table 6. Calculation of reagent feed concentrations at each pH value.

[0321] Furthermore, a regenerated cellulose-type ultrafiltration membrane with a 3 kilodalton molecular weight cutoff was used to confine the VHPO enzyme within the continuous flow reactor. A second peristaltic pump was with a UF setup (Cobetter, TFF, RC membrane, 0.11 m2, 3 kDa MWCO) to remove excess water. This was in a feedback loop with a mass scale to maintain a constant reactor volume. Simultaneously, an air bubbler system was used to continuously extract bromoform which was deposited onto a rotating drum of activated carbon granules. This was accomplished through a vapor deposition process where bromoform adsorbs onto a bed of activated carbon. A rotating drum was used to help maintain a homogeneous deposition. During the reaction, foaming could be concern and was addressed by the addition of a 300 ppm of silicone- based defoaming agent and a paddle stirrer at 500 rpm.

[0322] In summary, two control systems were used: a pump used to modulate flow through the filtration system to maintain a constant reactor volume (as indicated by a continuous real time mass measurement of the entire reactor and its content) and another pump used to add the reagents at rate necessary to maintain a constant pH. FIG. 8 and FIG. 9 show that after reaching the desired balance and pH setpoints the reagent pump and balance pump speeds could be modulated to maintain a near constant pH reading and balance reading, respectively. Initially, the pumps were operating at maximum speed to reach the setpoint value as quickly as possible. After reaching the setpoint value, the pH pump added reagents in a way that mimicked the reaction rate to maintain a constant pH. The balance pump was used to filter out excess water because of the reagent addition to maintain a constant volume.

[0323] In this example, the reactor continued for approximately 30 hours consuming approximately 10 L of reagent corresponding to approximately 40 g of dimedone. Based on the reaction stoichiometry and the molecular weight of the reagent, this corresponds to 72 grams of bromoform. GMCS results showed transfer of this bromoform onto the activated carbon carrier.

[0324] INCORPORATION BY REFERENCE

[0325] All references, articles, publications, patents, patent publications, and patent applications cited herein within the above text and / or cited below are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

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[0339] Roque, B. M., Brooke, C. G, Ladau, J., Polley, T., Marsh, L. J., Najafi, N., Pandey, P., Singh, L., Kinley, R., Salwen, J. K., Eloe-Fadrosh, E., Kebreab, E., & Hess, M. (2019). Effect of the macroalgae Asparagopsis taxiformis on methane production and rumen microbiome assemblage. Animal Microbiome, 1(1), 3.

[0340] Soedjak, H. S., Walker, J. V. & Butler, A. 1995. Inhibition and inactivation of vanadium bromoperoxidase by the substrate hydrogen peroxide and further mechanistic studies. Biochemistry, 34, 12689-12696.

[0341] Thapa, H. R., Lin, Z., Yi, D., Smith, J. E., Schmidt, E. W., & Agarwal, V. (2020). Genetic and Biochemical Reconstitution of Bromoform Biosynthesis in Asparagopsis Lends Insights into Seaweed Reactive Oxygen Species Enzymology. ACS Chemical Biology, 15(6), 1662-1670.

[0342] Wever, R., & Hemrika, W. (2001). Vanadium haloperoxidases. Handbook of Metalloproteins. Edited by Albrecht Messerschmidt, Robert Huber, Thomas Poulos and Karl Wieghardt. John Wiley & Sons, Ltd, Chichester.

[0343] Wood, J. M., Kennedy, F. Scott., & Wolfe, R. S. (1968). Reaction of multihalogenated hydrocarbons with free and bound reduced vitamin B12. Biochemistry, 7(5), 1707-1713.

[0344] Poulomi Majumdar et al., Chemistry of Dimedone for Synthesis of Oxygen-, Nitrogen-, and Sulfur- Containing Heterocycles from 2-(3-Hydroxy-5,5-dimethylcyclohex-2- enylidene)malononitrile, 43 Synth. Commun. 899-914 (2013)

Claims

CLAIMSWhat is claimed is:

1. A method for producing trihalomethane, comprising: adding at least one vanadium-dependent haloperoxidase (VHPO) enzyme to a solution in an reactor; adding one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV to the solutionadding hydrogen peroxide to the solution; and separating one or more trihalomethane produced by the at least one VHPO enzyme from the solution.

2. The method of claim 1, wherein in Formula II,R3, R4, Rs, Rs, R7, and Rs each independently represents hydrogen, halogen, hydroxyl, carboxyl, substituted or unsubstituted Ci-Ce alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or combinations thereof,69wherein one or more methylene groups of the substituted or unsubstituted Ci-Ce alkyl of R3, R4, R5, R6, R7, and / or R« may optionally be replaced by -O-, -NH-, -S-, -COO-, -COS-, CONH- , -CSNH-, and / or -CSO-.

3. The method of any one of claims 1 - 2, wherein the compound of Formula II is one or more of the following:

4. The method of claim 3, wherein the compound of Formula II is Formula Il-d.

5. The method of any one of claims 1 - 4, wherein the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV and the hydrogen peroxide are pre-mixed to form a mixture, the mixture is subsequently fed to the reactor.

6. The method of any one of claims 1 - 5, further comprising: adding at least one pH buffering agent to the solution.

7. The method of claim 6, wherein the at least one pH buffering agent is selected from the group consisting of: formic acid, acetic acid, propanoic acid, carbonic acid, fumaric acid, benzoic acid, oxalic acid, malonic acid, maleic acid, succinic acid, lactic acid, gluconic acid, adipic acid, o-toluic acid, benzene tetracarboxylic acid, citric acid, isocitric acid, aconitic acid, propane- 1,2, 3 -tricarboxylic acid, itaconic acid, citraconic acid, hydrogen chloride (HC1), sulfuric acid (H2SO4), nitric acid (HNO3), phosphoric acid (H3PO4), sodium hydroxide (NaOH), potassium hydroxide (KOH), monopotassium phosphate (KH2PO4), dipotassium phosphate (K2HPO4), N- cyclohexyl-2-aminoethanesulfonic acid (CHES), borate, sulfamic acid (H3NSO3), monosodium phosphate (NaH2PO4), disodium phosphate (Na2HPO4), sodium chloride (NaCl), potassium chloride (KC1), calcium chloride (CaCb), magnesium chloride (MgCh), ammonia,[tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), A,A-Bis(2- hydroxyethyl)glycine (Bicine), tris(hydroxymethyl)aminomethane (Tris), A-[ l ,3-Dihydroxy-2- (hydroxymethyl)propan-2-yl]glycine (Tricine), 3-[[l,3-dihydroxy-2-(hydroxymethyl)propan-2- yl] amino] -2-hydroxypropane-l -sulfonic acid (TAPSO), 2-[4-(2-Hydroxyethyl)piperazin-l- yl] ethane- 1 -sulfonic acid (HEPES), 2-{[l,3-Dihydroxy-2-(hydroxymethyl)propan-2- yl]amino} ethane- 1 -sulfonic acid (TES), 3-(V-morpholino)propanesulfonic acid (MOPS), piperazine- / / , V’ -bis(2-ethanesulfonic acid) (PIPES), and 2-(A-morpholino)ethanesulfonic acid (MES).

8. The method of any one of claims 6 - 7, wherein the at least one pH buffering agent is added to the mixture.

9. The method of any one of claims 1 - 8, further comprising: adding one or more halide salt to the solution, wherein the one or more halide salt is selected from the group consisting of: potassium chloride (KC1), potassium bromide (KBr), potassium iodide (KI), sodium chloride (NaCl), sodium bromide (NaBr), and sodium iodide (Nal).

10. The method of claim 9, wherein the one or more halide salt is added to the mixture.

11. The method of any one of claims 1 - 10, further comprising: immobilizing and / or confining the at least one VHPO enzyme to the reactor.

12. The method of claim 11 , wherein immobilization and / or confinement of the at least one VHPO enzyme to the reactor is accomplished by: coupling or attaching the at least one VHPO enzyme to a wall of the reactor, coupling or attaching the at least one VHPO enzyme to a plurality of beads, and / or confining the at least one VHPO enzyme with an ultrafiltration membrane.

13. The method of claim 12, wherein the at least one VHPO enzyme is coupled or attached to the wall of the reactor via covalent binding, affinity binding, and / or physical adsorption.7214. The method of claim 12, wherein the at least one VHPO enzyme is coupled or attached to the plurality of beads via covalent binding, affinity binding, and / or physical adsorption.

15. The method of claim 12, wherein the plurality of beads are made of a material comprising silica, magnetic materials, resins, polymers, and / or noble metals.

16. The method of claim 12, wherein the ultrafiltration membrane is made of a material comprising cellulose, hemicellulose, cellulose acetate, regenerated cellulose, polyether sulfone (PES), polysulfone (PSU), polyphenylene sulfone (PPSU), poly(bisphenol-A sulfone) (PSF), poly(arylene sulfone) (PAS), polylactide, poly(aryl ether ether ketone) (PEEK), polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET), polyethylene chlorotrifluoroethylene (PECTFE), polyethylene tetrafluoroethylene (PETFE), polytetrafluoroethylene (PTFE), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), polyvinylidene fluoride (PVDF), thermoplastic polyurethane (TPU), polyvinyl-fluoride, ethylenechlorotrifluoroethylene copolymer, ethylene-tetrafluoroethylene-copolymer, fluoroethylenepropylene copolymer, perfluoralkoxy polymer, and / or polychloro-trifluoroethylene.

17. The method of any one of claims 12 - 16, wherein the ultrafiltration membrane is installed in an ultrafiltration cassette.

18. The method of any one of claims 1 - 17, wherein the at least one VHPO enzyme, the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV, and the hydrogen peroxide are blended in the solution in the reactor.

19. The method of any one of claims 17 - 18, wherein the solution is filtered through the ultrafiltration cassette.

20. The method of any one of claims 18 - 19, wherein the solution is blended by an agitator blender, a paddle mixer, a dual shaft blender, a ribbon blender, a rotary blender, a vibrational blender, a static mixer, or a vertical blender.7321. The method of any one of claims 1 - 20, further comprising pumping an air flow into the solution.

22. The method of claim 21, further comprising directing the air flow to pass through the solution, such that the one or more trihalomethane produced by the at least one VHPO enzyme is separated from the solution and volatilized by the air flow.

23. The method of claim 22, further comprising directing the air flow to a tank, the tank contains one or more high surface area material to capture the one or more trihalomethane.

24. The method of claim 23, wherein the tank is a rotating drum.

25. The method of any one of claims 23 - 24, wherein the one or more high surface area material has a surface area of about 50 to about 5000 m2 / g, about 100 to about 5000 m2 / g, about 200 to about 5000 m2 / g, about 300 to about 5000 m2 / g, about 500 to about 5000 m2 / g, about 500 to about 4000 m2 / g, about 500 to about 3000 m2 / g, about 750 to about 3000 m2 / g, about 900 to about 3000 m2 / g, about 1000 to about 3000 m2 / g, or about 500 to about 1500 m2 / g.

26. The method of any one of claims 23 - 25, wherein the one or more high surface area material is a carbonaceous material, a hyper-crosslinked polymer, an aerogel, and / or a porous sorbent.

27. The method of claim 26, wherein the carbonaceous material is selected from the group consisting of: activated carbon (activated charcoal), graphene, graphene oxide, and derivatives thereof.

28. The method of any one of claims 23 - 27, wherein the one or more high surface area material is activated carbon.

29. The method of any one of claims 22 - 28, further comprising:74after directing the air flow to pass through the solution, directing the air flow to pass through at least one desiccant or water absorbing material.

30. The method of claim 29, wherein the at least one desiccant or water absorbing material is selected from the group consisting of: activated alumina, calcium chloride (CaCh), calcium oxide (CaO), calcium hydride (CaFT), calcium sulfate (CaSC ), cobalt(II) chloride (CoCb), copper(II) sulfate (Q1SO4), lithium chloride (LiCl), lithium bromide (LiBr), magnesium chloride (MgCb), magnesium sulfate (MgSC ), magnesium perchlorate (Mg(C104)2), Nafion, phosphorus pentoxide (P4O10), potassium carbonate (K2CO3), potassium hydroxide (KOH), sodium hydroxide (NaOH), silica gel, sodium, sodium chlorate (NaClOs), sodium chloride (NaCl), sodium sulfate (Na2SO4), sucrose, sulfuric acid (H2SO4), triethylene glycol, and a combination thereof.

31. The method of any one of claims 23 - 30, further comprising extracting the one or more trihalomethane from the one or more high surface area material.

32. The method of claim 31, wherein a hydrophobic solvent is applied to extract the one or more trihalomethane.

33. The method of any one of claims 1 - 32, wherein the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV, and / or the hydrogen peroxide are continuously added to the solution.

34. The method of any one of claims 9 - 10, wherein the one or more halide salt is continuously added to the solution.

35. The method of any one of claims 1 - 34, further comprising: adding one or more alkaline earth metal salt to the solution.

36. The method of claim 35, wherein the one or more alkaline earth metal salt comprises: calcium oxide (CaO), calcium acetate, calcium carbonate (CaCO-,), calcium chloride(CaCb), calcium citrate, calcium gluconate, calcium sulfate (CaSC ), calcium bromide (CaBn), calcium hydroxide (Ca(OH)2), calcium nitrate (Ca(N0s)2), magnesium oxide (MgO), magnesium acetate, magnesium carbonate (MgCCh), magnesium chloride (MgCb), magnesium citrate, magnesium gluconate, magnesium sulfate (MgSC ), magnesium bromide (MgBn), magnesium hydroxide (Mg(0H)2), magnesium nitrate (Mg(NOs)2), and / or a combination thereof.

37. The method of any one of claims 1 - 36, wherein the at least one VHPO enzyme is recombinantly expressed by a fungal strain.

38. The method of claim 37, wherein the fungal strain is a yeast belonging to the genus of Arxula, Candida, Ogataea, Kluyveromyces, Pichia, Saccharomyces, or Yarrowia.

39. The method of claim 38, wherein the yeast is Saccharomyces cerevisiae, Yarrowia lipolytica, or Pichia pastoris.

40. The method of claim 37, wherein the fungal strain is a filamentous fungus.

41. The method of claim 40, wherein the filamentous fungus belongs to a genus selected from the group consisting of Altemaria, Ascomycota, Bipolaris, Cadophora, Cladorrhinum, Clohesyomyces, Curvularia, Darksidea, Didymella, Dothideomycetes, Drechslera, Exserohilum, Epicoccum, Exophiala, Gaeumannomyces, Gliomastix, Helotiales, Keissleriella, Laburnicola, Lachnum, Leptodontidium, Magnaporthiopsis, Mollisia, Neocamarosporium, Oidiodendron, Ophiosphaerella, Penicillium, Phaeosphaeria, Phialophora, Phoma, Pleosporales, Poaceascoma, Septoriella, Slopeiomyces, Sordariales, Trametes, Trematosphaeria, Trichoderma, and Tricladium.

42. The method of claim 41, wherein the filamentous fungus belongs to a species selected from the group consisting of Altemaria chlamydospora, Cladorrhinum foecundissimum, Curvularia inaequalis, Curvularia spicifera, Curvularia intermedia, Curvularia pseudobrachyspora, Curvularia hawaiiensis, Curvularia protuberata, Curvularia lunata, Darksidea alpha, Darksidea zeta, Leptodontidium orchidicola, Magnaporthiopsis panicorum,Mollisia cinerea, Neocamarosporium phragmitis, Oidiodendron citrinum, Penicillium penarojense, Penicillium griseolum, Phaeosphaeria caricis, Phaeosphaeria microscopica, Phaeosphaeria poagena, Phaeosphaeria sinensis, Poaceascoma lochia, Septoriella leuchtmannii, Slopeiomyces cylindrosporus, Trametes versicolor, Trematosphaeria hydrela, Trematosphaeria terricola, Trichoderma lixii, and Tricladium terrestre.

43. The method of any one of claims 1 - 42, wherein the trihalomethane is selected from the group consisting of chloroform, bromoform, iodoform, bromodichloromethane, dibromochloromethane, and combinations thereof.

44. The method of any one of claims 1 - 43, wherein the trihalomethane is bromoform.

45. The method of any one of claims 1 - 44, wherein the solution has a pH value of between about 4.0 to about 10.0, between about 4.0 to about 9.5, between about 4.0 to about 9.0, between about 4.0 to about 8.5, between about 4.0 to about 8.0, between about 4.0 to about 7.5, between about 4.0 to about 7.0, between about 4.0 to about 6.75, between about 4.0 to about 6.5, between about 4.25 to about 6.5, between about 4.5 to about 6.5, between about 4.75 to about 6.5, between about 5.0 to about 6.5, between about 5.0 to about 6.25, or between about 5.0 to about 6.0.

46. A device for producing trihalomethane, comprising: a reactor, wherein a solution and at least one vanadium-dependent haloperoxidase (VHPO) enzyme are disposed in the reactor; a first reservoir, the first reservoir is connected to the reactor and contains one or more compounds of Formula I, Formula II, Formula III, and / or Formula IVa second reservoir, the second reservoir is connected to the reactor and contains hydrogen peroxide; and a tank, the tank is connected to the reactor and contains one or more high surface area material.

47. The device of claim 46, wherein the first reservoir transfers the one or more compounds of Formula I, Formula II, FormulaIII, and / or Formula IV to the reactor; and the second reservoir transfers the hydrogen peroxide to the reactor.

48. The device of any one of claims 46 - 47, further comprising: a third reservoir, the third reservoir is connected to the first reservoir, the second reservoir, and the reactor, wherein the third reservoir receives the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV transferred from the first reservoir and the hydrogen peroxide transferred from the second reservoir, such that the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV and the hydrogen peroxide are pre-mixed to form a mixture, wherein the third reservoir transfers the mixture to the reactor.

49. The device of any one of claims 46 - 48, further comprising: a fourth reservoir, the fourth reservoir is connected to the reactor and contains at least one pH buffering agent,78wherein the fourth reservoir transfers the at least one pH buffering agent to the reactor.

50. The device of claim 49, wherein the fourth reservoir transfers the at least one pH buffering agent to the third reservoir.

51. The device of any one of claims 46 - 50, further comprising: an air pump, the air pump pumps an air flow into the solution.

52. The device of any one of claims 46 - 51, further comprising: a fifth reservoir, the fifth reservoir is connected to the reactor and the tank, the fifth reservoir contains at least one desiccant or water absorbing material, wherein the air flow is directed to flow from the reactor to the tank through the fifth reservoir, such that a moisture contained with the air flow is removed by the at least one desiccant or water absorbing material.

53. The device of any one of claims 46 - 52, wherein one or more trihalomethane is produced by the at least one VHPO enzyme in the reactor, wherein the one or more trihalomethane is captured by the one or more high surface area material in the tank.

54. The device of any one of claims 46 - 53, further comprising: an ultrafiltration cassette, the ultrafiltration cassette is connected to the reactor and contains an ultrafiltration membrane to confine the at least one VHPO enzyme in the reactor, wherein the solution flows from the reactor to the ultrafiltration cassette such that the solution is filtered through the ultrafiltration membrane, and the solution flows from ultrafiltration cassette back to the reactor.

55. The device of claim 54, wherein the ultrafiltration membrane is made of a material comprising cellulose, hemicellulose, cellulose acetate, regenerated cellulose, polyether sulfone (PES), polysulfone (PSU), polyphenylene sulfone (PPSU), poly(bisphenol-A sulfone) (PSF), poly(arylene sulfone) (PAS), polylactide, poly(aryl ether ether ketone) (PEEK), polypropylene79(PP), polyethylene (PE), polyethylene terephthalate (PET), polyethylene chlorotrifluoroethylene (PECTFE), polyethylene tetrafluoroethylene (PETFE), polytetrafluoroethylene (PTFE), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), polyvinylidene fluoride (PVDF), thermoplastic polyurethane (TPU), polyvinyl-fluoride, ethylenechlorotrifluoroethylene copolymer, ethylene-tetrafluoroethylene-copolymer, fluoroethylenepropylene copolymer, perfluoralkoxy polymer, and / or polychloro-trifluoroethylene.

56. The device of any one of claims 46 - 55, wherein the at least one VHPO enzyme is immobilized and / or confined in the reactor.

57. The device of claim 56, wherein immobilization and / or confinement of the at least one VHPO enzyme to the reactor is accomplished by: coupling or attaching the at least one VHPO enzyme to a wall of the reactor, coupling or attaching the at least one VHPO enzyme to a plurality of beads, and / or confining the at least one VHPO enzyme with an ultrafiltration membrane.

58. The device of claim 57, wherein the at least one VHPO enzyme is coupled or attached to the wall of the reactor via covalent binding, affinity binding, and / or physical adsorption.

59. The device of claim 57, wherein the at least one VHPO enzyme is coupled or attached to the plurality of beads via covalent binding, affinity binding, and / or physical adsorption.

60. The device of claim 57, wherein the plurality of beads are made of a material comprising silica, magnetic materials, resins, polymers, and / or noble metals.

61. The device of any one of claims 46 - 60, wherein the first reservoir further contains one or more halide salt.

62. The device of any one of claims 46 - 61, further comprising a sixth reservoir, the sixth reservoir is connected to the reactor and contains one or more alkaline earth metal salt.8063. The device of any one of claims 46 - 62, wherein the first reservoir, the second reservoir, the third reservoir, the fourth reservoir, and / or the sixth reservoir continuously transfer the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV, the hydrogen peroxide, the at least one pH buffering agent, the one or more halide salt, and / or the one or more alkaline earth metal salt to the reactor.

64. The device of any one of claims 46 - 63, wherein the reactor comprises an agitator blender, a paddle mixer, a dual shaft blender, a ribbon blender, a rotary blender, a vibrational blender, a static mixer, and / or a vertical blender, and wherein the solution is blended in the reactor.

65. The device of any one of claims 46 - 64, wherein the tank is a rotating drum.

66. The device of any one of claims 46 - 65, wherein transfer of the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV and / or the one or more halide salt from the first reservoir to the reactor is facilitated by a first pump.

67. The device of any one of claims 46 - 66, wherein transfer of the hydrogen peroxide from the second reservoir to the reactor is facilitated by a second pump.

68. The device of any one of claims 54 - 67, wherein transfer of the solution from the reactor to the ultrafiltration cassette and return from the ultrafiltration cassette to the reactor is facilitated by a third pump.

69. The device of any one of claims 49 - 68, wherein transfer of the at least one pH buffering agent from the fourth reservoir to the reactor is facilitated by a fourth pump.

70. A device for producing trihalomethane, comprising: a reactor, wherein a solution and at least one vanadium-dependent haloperoxidase (VHPO) enzyme are disposed in the reactor;81a first reservoir, the first reservoir contains one or more compounds of Formula I, Formula II, Formula III, and / or Formula IVFormula III Formula IV a second reservoir, the second reservoir contains hydrogen peroxide; a third reservoir; a fourth reservoir, the fourth reservoir contains at least one pH buffering agent, wherein the third reservoir is connected to the first reservoir, the second reservoir, the fourth reservoir, and the reactor, wherein the first reservoir transfers the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV to the third reservoir, the second reservoir transfers the hydrogen peroxide to the third reservoir, and the fourth reservoir transfers the at least one pH buffering agent to the third reservoir, such that the one or more compounds of Formula I, Formula II, Formula III, and / or Formula IV, the hydrogen peroxide, and the at least one pH buffering agent are pre-mixed to form a mixture, wherein the third reservoir transfers the mixture to the reactor; an air pump, the air pump pumps an air flow into the solution;82an ultrafiltration cassette, the ultrafiltration cassette is connected to the reactor and contains an ultrafiltration membrane to confine the at least one VHPO enzyme in the reactor, wherein the solution flows from the reactor to the ultrafiltration cassette such that the solution is filtered through the ultrafiltration membrane, and the solution flows from ultrafiltration cassette back to the reactor; and a tank, the tank is connected to the reactor and contains one or more high surface area material, wherein the air flow is directed to flow from the reactor to the tank, wherein one or more trihalomethane is produced by the at least one VHPO enzyme in the reactor, wherein the one or more trihalomethane is captured by the one or more high surface area material in the tank.83

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