Degradation method

Abstract

DEGRADATION METHOD. The present invention relates to a method of degrading biofilm by contacting it with an aqueous mixture comprising a peroxide compound and a manganese complex, wherein the aqueous mixture comprises a macrocyclic ligand. The invention also relates to a method of degrading a biofilm by contacting it with an aqueous mixture comprising a peroxide compound and a macrocyclic ligand.

Description

1 / 99 Descriptive Report of Invention Patent: DEGRADATION METHOD Field of Invention

[0001] The present invention relates to a method of degrading biofilm by contacting it with an aqueous mixture comprising a peroxide compound and a manganese complex, wherein the aqueous mixture comprises a macrocyclic ligand. The invention also relates to a method of degrading a biofilm by contacting it with an aqueous mixture comprising a peroxide compound and a macrocyclic ligand. Background of the Invention

[0002] Biofilms are defined by M. Vert et al. in Pure Appl. Chem., 2012, 84 (2), 377-410 as aggregates of microorganisms in which the cells, which are often embedded within a self-produced matrix of extracellular polymeric substances (EPS), adhere to each other and / or to a surface. EPS are produced by microorganisms within the matrix and typically comprise polysaccharides such as alginate, murein, colonic acid, bacterial cellulose, dextran, kefiran, curdlan, welan, gellan, and xanthan (see, for example, B. Vu et al in Molecules 2009, 14, 2535-2554). Because biofilms generally require water to form, they are especially common in equipment that is frequently or permanently exposed to aqueous environments, i.e., equipment operated in the presence of water. Biofilms are frequently found on membranes present in all types of filtration devices.All of these membranes are susceptible to fouling with biofilms, particularly those found in reverse osmosis systems.

[0003] In addition to membranes, other equipment may also be susceptible to biofilm formation, in particular, equipment that is frequently exposed to aqueous environments. This includes pipes and plumbing equipment; cleaning equipment (including laundry, Petition 870220076624, dated 08 / 25 / 2022, page 5 / 115 2 / 99 dishwashing and bathing), such as sinks, bathtubs, showers, dishwashers, washing machines, dryers, bidets and surfaces within spaces used for cleaning (e.g., shower walls and floors); refrigeration and heating systems; water vessels (including ship hulls and boats); and marine equipment. Aqueous mixtures are commonly used in oil and gas operations, and biofilm formation, for example, in pipelines and other production equipment, is a significant problem (see, for example, D. Xu and T. Gu in J. Microb. Biochem. Technol. 2015, 7(5)).

[0004] Biofilms can restrict or block flow through equipment and can corrode materials, thus reducing the lifespan of the material. Furthermore, biofilms sometimes contain pathogens, such as Legionella, which can cause damage when present in water sources. Preventing and / or treating biofilms reduces the need for equipment maintenance and cleaning, and therefore can lead to lower system maintenance and operating costs. Thus, biofilm degradation and prevention is commercially viable. However, biofilm degradation can be challenging.

[0005] Biofilms can restrict or block flow through equipment and can corrode materials, thus reducing the lifespan of the material. Furthermore, biofilms sometimes contain pathogens, such as Legionella, which can cause damage when present in water sources. Preventing and / or treating biofilms reduces the need for equipment maintenance and cleaning, and therefore can lead to lower system maintenance and operating costs. Thus, biofilm degradation and prevention is commercially viable. However, biofilm degradation can be challenging.

[0006] Biofilms possess several defense mechanisms: EPS can act as a diffusion barrier for degrading materials, and the cells within biofilms are able to decrease their metabolism in the presence of degrading materials, use efflux pumps to remove degrading materials from within the cell, and multiply rapidly upon removal of the degrading material, thus allowing for rapid recovery of the biofilm afterward. Petition 870220076624, dated 08 / 25 / 2022, page 6 / 115 3 / 99 exposure to degrading materials (see D. Xu, and T. Gu, above).

[0007] Biofilm EPS generally contains polysaccharides or proteins. Consequently, degradation methods often involve the addition of proteases and / or the addition of amylases, see, for example, IP Molobela, TE Cloete and M. Beukes, African J. of Microbiology Research, 2010, 4 (14), 1515-1524. Since biofilms comprise colonies of microorganisms, antimicrobial agents can be employed in degradation methods. However, it has been found that the antimicrobial resistance of microorganisms within biofilms is greater than that of planktonic microorganisms, which may render biofilm degradation methods using antimicrobials ineffective (see R. Patel, Clin. Orthop. Relat. Res., 2005, 437, 41-47).

[0008] Biofilms can be removed from equipment using cleaning solutions comprising organic peroxyacids (see documents WO 2019 / 160948 A1 and WO 2017 / 181005 A1, both Ecolab USA Inc.), or surfactants and enzymes (see document WO 03 / 022752 A1, Advanced Biocatalytics Corp.).

[0009] Alternatively, biofilm formation can be avoided by using copolymers to reduce the adhesion of microorganisms to surfaces of interest (see, for example, WO 2009 / 071451 A2 (Henkel AG & CO KGaA)), by using compositions comprising cyclic ketones (see WO 2018 / 009076 A1 (Inhibio AS)), or by using ultrasonic waves (see WO 2019 / 159021 A1, Harteel BVPA).

[0010] Biofilms commonly found generally comprise alginic acids or alginates (used similarly here). Alginates are hydrophilic polysaccharides and are commonly found in the cell walls of brown algae and various other microorganisms.

[0011] The use of iron salts and hydrogen peroxide to degrade alginates was described by O Smidsrod et al. in Acta Chem. Scand., 1965, 19, 143-152. It is shown that preferentially high levels (>0.1 M) of peroxide Petition 870220076624, dated 08 / 25 / 2022, p. 7 / 115 4 / 99 hydrogen and iron(III) chloride (>100 μM) lead to a reduction in the viscosity of alginates, attributed to the formation and involvement of hydroxyl radicals.

[0012] In document WO 2018 / 115867 A2 (Marine Biopolymers Ltd.), a process is described for obtaining a target chemical species from seaweed, which includes a bleaching step of a portion of seaweed. The bleaching step comprises the use of a bleaching composition, which may include an oxidation catalyst, which may be [(MnIV)2(pO)3(Me3-TACN)2]2+, [(MnIII)2(pO)(p-CH3COO)2(Me3-TACN)2]2+, or [MnIIIMnIV(pO)2(p-CH3COO)(Me4-DTNE)]2+; or suitable salts thereof. The subsequent optional depolymerization of the alginate or salt thereof is also described, but the use of manganese catalysts to degrade or depolymerize polymers is not disclosed in this application.

[0013] It would be beneficial to develop at least alternative methods for degrading biofilms, for example, by degrading and / or depolymerizing EPS and / or materials within the microorganisms of the biofilms (including materials such as alginates within the cell walls of the microorganisms). The present invention addresses this issue. Summary of the Invention

[0014] The present invention is based on the discovery that aqueous mixtures comprising peroxide compounds, manganese complexes, and macrocyclic ligands are surprisingly active in degrading biofilms. The various defense mechanisms exhibited by biofilms to inhibit their degradation make them more difficult to degrade compared to their constituent materials, and the use of the aqueous mixtures described herein for degrading biofilms has not been previously disclosed.

[0015] Viewed from a first aspect, therefore, the present invention provides a method for degrading a biofilm comprising contacting the biofilm with an aqueous mixture comprising (i) a peroxide compound and (ii) a mononuclear manganese complex of Mn(II), Mn(III) or Mn(IV), or a binuclear complex of Mn(II)Mn(II), Mn(III)Mn(II), Mn(III)Mn(III), Mn(III)Mn(IV) or Petition 870220076624, dated 08 / 25 / 2022, page 8 / 115 5 / 99 Mn(IV)Mn(IV), wherein the aqueous mixture comprises a ligand of formula (I) or (II): (Q)p CR-ι R2 (I) CR3R4 (II),

[0016] where: ---N--- [CR1R2CR3R4)

[0017] p is 3;

[0018] each R is independently selected from the group consisting of hydrogen, C1-C24 alkyl, CH2Ce-C1 caryl, CH2CH2OH, CH2COOH, and pyridin-2-ylmethyl;

[0019] Q' is an ethylene or propylene bridge; and

[0020] R1, R2, R3 and R4 are independently selected from: H, C1-C4 alkyl and C1-C4 hydroxyalkyl.

[0021] Biofilms generally contain manganese ions which, in addition to macrocyclic ligands, form manganese complexes that are active in biofilm degradation in the presence of a peroxide compound. Thus, viewed from a second aspect, the present invention provides a method for degrading a biofilm comprising contacting the biofilm with an aqueous mixture comprising a peroxide compound and a ligand as defined according to the first aspect. Petition 870220076624, dated 08 / 25 / 2022, page 9 / 115 6 / 99

[0022] Additional aspects and realizations of the present invention will become apparent from the discussion that follows below. Brief Description of the Figure

[0023] Figure 1 shows a graph of the specific viscosity of alginate divided by the concentration of alginate in solution as a function of the alginate concentration. The intrinsic viscosity can be determined from the Y-intercept of the graph, as described in more detail in the experimental part section below. Detailed Description of the Invention

[0024] In the following discussion, reference is made to various terms, which have the meanings given below, unless a context indicates otherwise. The nomenclature used here to define compounds, in particular the compounds according to the invention, is generally based on the IUPAC organization rules for chemical compounds, specifically the “IUPAC Compendium of Chemical Terminology (Gold Book)”. For the avoidance of doubt, if an IUPAC organization rule conflicts with a definition given in this document, the definition in this document shall prevail. Furthermore, if a compound structure conflicts with the name given for the structure, the structure shall prevail.

[0025] The term comprising or variants thereof shall be understood here as implying the inclusion of a declared element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0026] The term consisting or variants thereof shall be understood here as implying the inclusion of a declared element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0027] The term about in this document, when qualifying a number or value, is used to refer to values ​​that lie within ± 5% of the specified value. For example, if a molar ratio of formula (I) for manganese Petition 870220076624, dated 08 / 25 / 2022, page 10 / 115 7 / 99 for molar ratios of approximately 100:1 to approximately 0.1:1, including molar ratios of 105:1 to 0.095:1.

[0028] References to physical states of matter (such as liquid or solid) refer to the state of matter at 25 °C and atmospheric pressure, unless the context specifies otherwise.

[0029] As summarized above, the present invention is based on the surprising discovery that contact of a biofilm with an aqueous mixture comprising a peroxide compound, a manganese complex and a macrocyclic ligand of formula (I) or (II) results in the degradation of the biofilm.

[0030] Biofilm degradation, herein, should be understood as the breakdown of at least a portion of the molecular structure of the biofilm by the cleavage of molecular bonds. It should be understood that degradation is not limited to the complete destruction of the molecular structure of the biofilm. Partial degradation of the molecular structure of the biofilm is included. Biofilm degradation can be achieved, for example, by depolymerizing polymeric substances within the biofilm. Degradation can be measured in several ways (see Wilson, C. et al., Res. Rev. J. Eng. Technol., 2017, 6(4), 1-42; and Paquet-Mercier, F. et al., Lab Chip., 2016, 16(24), 4710-4717).It can be measured as a loss of biofilm mass, for example, the dry mass or total carbon content of the biofilm; a reduction in the viscosity of the biofilm or in the viscosity of a component within the biofilm, for example, the dynamic viscosity of alginate within the biofilm; or a change in the color or absorbency of the biofilm itself, or the biofilm staining with a suitable dye.

[0031] Viscosity is a measure of the internal friction of (in this document) a fluid. The dynamic viscosity of a fluid expresses its resistance to shear forces when adjacent layers move parallel to each other at different speeds. In any viscosity measurement of a (bio)polymer, it is important to maintain a constant solution temperature and employ a constant weight percentage of the (bio)polymer in aqueous solutions, as these parameters affect the viscosities of said solutions. In Petition 870220076624, dated 08 / 25 / 2022, page 11 / 115 8 / 99 Vauchel et al., J. Phycol. 2008, 44, 515-517, dynamic viscosity and capillary viscosity measurements of alginates are used to derive the average polymer chain lengths of alginate. The average polymer chain lengths of alginates became shorter as the alkaline extraction time increased, i.e., the degree of degradation increased. Thus, dynamic viscosity measurements were employed to obtain information on the degree of alginate degradation.

[0032] Biofilm degradation can be measured by evaluating the reduction in dynamic viscosity of the components within the biofilm after degradation and comparing it with the dynamic viscosity of the same components before degradation. For example, alginate may be present in the EPS of the biofilm. The dynamic viscosity of the alginate extracted from the biofilm before and after degradation can be used to give an indication of the degree of biofilm degradation. To evaluate the dynamic viscosity of alginate, it is extracted from the biofilm. J. Wingender, et al., Methods Enzymol., 2001, 336, 302-314 describe a method for extracting alginate from biofilm.After separating the microorganism cells from the EPS by centrifugation and dialysis (to remove low molecular weight matter), the polysaccharides can be isolated from the remaining material by adding an organic solvent and treating with nucleases and proteases (to degrade the nucleic acid and protein components, leaving the polysaccharide components intact). When bound to cations, specifically medications like Ca2+, alginates can be difficult to handle. By adding an acid, the carboxylate groups become protonated and form gelled alginic acid. This material can be converted into the sodium alginate salt, filtered, and subsequently purified by adding a calcium salt, giving a precipitation of Ca alginate, or by adding a strong acid to isolate the alginic acid.

[0033] In some embodiments, the method of the invention reduces the dynamic viscosity of the alginate within the biofilm by at least about 10%, preferably by at least about 20% (where the dynamic viscosity Petition 870220076624, dated 08 / 25 / 2022, p. 12 / 115 9 / 99 is typically measured at 25 °C.

[0034] Alternatively, the difference in dry mass (typically given as mass per unit area) of the biofilm, before and after being degraded using the methods of the invention, can be used to assess the degree of biofilm degradation. Portions of the biofilm's molecular structure are cleaved during degradation and can be easily separated from the residual biofilm mass, for example, by washing the biofilm with water or another liquid. The residual biofilm dry mass is found by placing the biomass in an oven at an elevated temperature (e.g., about 60 °C to about 105 °C), without decomposition (and therefore mass loss) of the biofilm, until the water has been removed. This occurs when the biofilm mass is constant with heating time. The resulting mass value is divided by the area that was covered by the biomass sample (before drying) to obtain the dry mass as mass per unit area.

[0035] In some embodiments, the methods of the invention reduce the dry mass of the biofilm by at least about 1% by weight, preferably by at least about 10% by weight.

[0036] A commonly used method for the detection and quantitative analysis of biofilms is described by C. Larimer et al. in Analytical and Bioanalytical Chem., 2016, 408, 999. In this method, broad-spectrum biomolecular staining is used to enhance the visibility of cells, nucleic acids, and proteins within the biofilm. The amount of biofilm is then quantitatively determined by digital image analysis, based on the intensity of the biofilm color after staining. Alternatively, cationic dyes, such as Crystal Violet, can be used. These dyes adhere to anionic polysaccharides and other negatively charged constituents of biofilms. Crystal Violet assays are frequently used to assess the amount of biofilm present. The use of such assays to assess the amount of biofilm in microtiter plates using microplate readers is described by E. Burton et al. in J. Ind. Microbiol. Biotechnol., 2007, 34(1), 1-4. In the same. Petition 870220076624, dated 08 / 25 / 2022, p. 13 / 115 In article 10 / 99, spectrofluorometric assays are also described, which are based on the binding of fluorescent probes to N-acetylglucosamide present in biofilms. The amount of biofilm is then measured quantitatively using a fluorescent plate reader.

[0037] Biofilms, as defined in this document, are aggregates of microorganisms in which the cells, which are frequently embedded within a self-produced matrix of extracellular polymeric substances (EPS), adhere to each other and / or to a surface (see M. Vert et al., supra). EPS are produced by microorganisms within the matrix and typically comprise polysaccharides such as alginate, murein, collagen, bacterial cellulose, dextran, kefiran, curdlan, welan, gellan, and xanthan gum (see B. Vu et al. in Molecules 2009, 14, 2535-2554 and Sutherland, IW, Microbiology, 2001, 147, 3-9).

[0038] Consistent with its use in the art, and as reviewed by K. Yong Lee and DJ Mooney, Prog. Polym. Sci. 2012, 37(1), 106-126, the terms alginate(s) and alginic acid(s) are used interchangeably herein to refer to linear copolymers composed of 1,4-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) building blocks. The monomers may appear in homopolymeric blocks of G residues (G blocks, as a block with the structure: GGGGGGG), M residues (M blocks, as a block with the structure: MMMMMM) or alternating M and G residues (MG blocks, as a block with the structure: MGMGMGMGMG). Alginates may comprise any number of combinations of M blocks, G blocks and / or MG blocks.The solubility of alginate in water is high at pH values ​​above the pKa values ​​of β-D-mannuronic acid (3.38) and α-L-guluronic acid (3.65), but at pH values ​​below the pKa values, an increasing extent of acid groups become protonated, reducing the polymer's solubility. Polymers with MG blocks have higher water solubility than polymers with M and G blocks. The ratio of M to G within an alginate and the length of the blocks within an alginate differ depending on the alginate source. Petition 870220076624, dated 08 / 25 / 2022, page 14 / 115 11 / 99

[0039] Biofilms can comprise alginate from different bacterial genera. Examples include the genera Azotobacter and Pseudomonas, specifically from bacterial cell walls. Alginate is often an important component of a biofilm matrix.

[0040] Biofilms can include murein, which is frequently found within the walls of bacterial cells. Because Gram-positive bacteria are characterized by thicker cell walls (compared to Gram-negative bacteria), they are typically sources of murein. Biofilms frequently comprise murein from different bacteria, typically Gram-positive bacteria, such as bacteria of the genus Staphylococcus, for example, Staphylococcus epidermidis. Murein is a peptidoglycan formed by sugars and amino acids. The sugar consists of alternating β-(1,4)-linked N-acetylglucosamine and N-acetylmuramic acid. Each N-acetylmuramic acid residue is linked to a chain of 4 or 5 amino acids through its lactic acid residue. The amino acid chain comprises a combination of amino acids, which can be L- or denantiomers. Examples include alanine, glutamic acid, glutamine, lysine, glycine, and mesodiaminopimelic acid.The amino acid chain can cross-link with another amino acid chain within the murein.

[0041] Biofilms can comprise colonic acids, which can be produced by bacteria, such as bacteria of the Enterobacteriaceae family, for example, bacteria of the genera Enterobacter and Klebsiella. Colonic acids are branched polysaccharides comprising glucose, galactose, fucose, glucuronic acid, acetate, and pyruvate.

[0042] Biofilms can comprise bacterial cellulose, from bacterial genera such as Acetobacter, Sarcina ventriculi, and Agrobacterium. Bacterial cellulose refers to polymers composed of β1,4-linked D-glucose units produced by bacteria. It has significantly different macromolecular properties from plant cellulose. For example, bacterial cellulose is typically more chemically pure, has greater hydrophilicity, and Petition 870220076624, dated 08 / 25 / 2022, page 15 / 115 12 / 99 greater tensile strength. Bacterial cellulose is typically produced as an extracellular polysaccharide that forms a protective barrier around the bacteria.

[0043] Biofilms may comprise dextran, which can be produced by bacteria, such as bacteria of the genus Streptococcus. Dextran refers to microbial-derived poly-αD-glucosides with α-1,6 glycosidic linkages. Dextran is a branched polysaccharide with branching α-1,3 glycosidic linkages. The specific structure of dextran depends on the strain of the microbe from which it is produced.

[0044] Biofilms may comprise kefiran, which can be produced by bacteria such as the genus Lactobacillus. Kefiran is a branched polysaccharide composed of approximately equal proportions of glucose and galactose. As reported by Ghasemlou, M. et al in Food Chem., 2012, 133(2), 383389, kefiran has a backbone of glucose linked to (1^6), galactose linked to (1^3), (1 galactose linked to 4), glucose linked to 1 4 and galactose linked to 1 2,6 (with an O-2 linked branch of galactose residues and terminated with glucose residues).

[0045] Biofilms may comprise curdlan, which can be produced from bacteria, such as bacteria of the genus Agrobacterium. Curdlan is a linear β-1,3-glucan, comprising entirely 1,3-ε-D-glycosidic linkages. It can be produced as an exopolysaccharide by bacteria, such as bacteria of the genus Agrobacterium.

[0046] Biofilms may comprise welan, which can be produced by bacteria, such as bacteria of the genus Alcaligenes. Welan is a branched polysaccharide consisting of repeating tetrasaccharide units comprising two monomers of D-glucose, D-glucuronic acid and L-rhamnose, with monomeric side chains of L-rhamnose or L-mannose at C3 of each glucose linked to 1,4.

[0047] Biofilms can comprise gellan, which can be produced by bacteria, such as bacteria of the genus Sphingomonas. Gellan is similar in Petition 870220076624, dated 08 / 25 / 2022, p. 16 / 115 13 / 99 has a Welan structure, but it is a linear polysaccharide, meaning it does not include L-rhamnose or L-mannose side chains.

[0048] Biofilms may comprise xanthan gum, which can be produced by bacteria, such as bacteria of the genus Xanthomonas. Xanthan gum refers to a branched polysaccharide, with a backbone consisting of ε-(1,4)-D-glucose. Each alternate glucose residue is linked to a three-sugar side chain consisting of a glucuronic acid residue positioned between two mannose residues. An acetyl group may be attached to the C6 position of the mannose residue positioned closest to the backbone, and a pyruvate group may be attached to the C4 and C6 positions of the terminal mannose.

[0049] Without being limited by theory, the methods of the invention are understood to depolymerize the polysaccharides within the cell walls of microorganisms within the biofilm and / or within the EPS of the biofilm, thereby breaking down the cell walls of the microorganisms and / or breaking down the EPS, degrading the biofilm and allowing for easier removal of the biofilm by conventional cleaning processes.

[0050] The methods of the invention comprise contacting the biofilm with an aqueous mixture. It will be understood that contact can be achieved in various ways. Preferably, however, the aqueous mixture is applied to the biofilm or to a mixture comprising the biofilm. The application method can be any method that results in contact between the aqueous mixture and the biofilm. Typically, the aqueous mixture is applied to the biofilm as a solution, a foam, or a suspension, preferably a solution or a foam. The aqueous mixture can be applied to the biofilm by squirting or spraying onto the biofilm. Alternatively, a mixture comprising the biofilm can be applied to the aqueous mixture, or the biofilm can be applied directly to the aqueous mixture.

[0051] Mixture is used in this document to refer to a combination of two or more components. For example, the aqueous mixture of the first aspect of the invention comprises water, a peroxide compound, a complex Petition 870220076624, dated 08 / 25 / 2022, p. 17 / 115 14 / 99 of manganese and a macrocyclic ligand. The aqueous mixture may be a suspension (e.g., a paste or slurry), comprising a solution in which a proportion of the peroxide compound, manganese complex, and macrocyclic ligand of formula (I) or (II) is dissolved, with the remaining proportion suspended in solution. Alternatively, the aqueous mixture may be a solution in which the peroxide compound, the manganese complex, and the macrocyclic ligand are dissolved. The aqueous mixture is typically a solution.

[0052] If a solution, the aqueous mixture of the first aspect of the invention is typically formed by dissolving the manganese complex, peroxide compound and (if added separately to the manganese complex) ligand of formula (I) or (II) in a (optionally buffered) solvent (typically water). It should be understood that any suitable method may be used to form the aqueous mixtures of the invention, the following description focusing on the first aspect of the invention (those skilled in the art will understand that appropriate adjustments (e.g., to omit a manganese complex) may be made with respect to the method of the second aspect of the invention in which the aqueous mixture need not comprise a manganese complex). The peroxide compound may be commercially available as a solution and may be added as a solution to the solvent before, after or with (i.e., at the same time as) addition of the manganese complex and optional ligand.For example, an aqueous solution comprising a peroxide compound may be added to water before, after, or with the manganese complex, which may be [MnIVMnIV(pO)3(1,4,7-trimethyl-1,4,7-triazacyclononane)2][CH3COO]2 (“μ” denoting, according to convention, a bridging ligand). Alternatively, if the peroxide compound is commercially available as a solid, it may be dissolved in a (optionally buffered) solvent (typically water) before coming into contact with the manganese complex and the optional ligand. Examples of solid peroxide compounds include sodium percarbonate, sodium perborate monohydrate, and sodium perborate tetrahydrate. Petition 870220076624, dated 08 / 25 / 2022, p. 18 / 115 15 / 99

[0053] If the ligand of formula (I) or (II) is added separately to the manganese complex, it may be added to the solvent before, after, or with the manganese complex, and the peroxide compound may be added similarly before, after, or with the ligand. For example, the ligand, such as 1,1,4,7-trimethyl-1,4,7-triazacyclononane, may be added to the water before, after, or at the same time as the addition of the manganese complex and the addition of an aqueous solution comprising the peroxide compound. If the peroxide compound is a solid, it may be mixed with the manganese complex and the optional ligand of formula (I) or (II) (if it is not part of the manganese complex). The resulting mixture may then be added to the solvent and dissolved to form the aqueous mixture. For example, a mixture of solid sodium percarbonate, manganese complex, and a ligand such as 1,4,7-trimethyl-1,4,7-triazacyclononane can be added to water and dissolved in the water.Normally, however, the solid peroxide compound is first dissolved in water, to which the manganese complex and the optional ligand of formula (I) or (II) (if not part of the manganese complex) are added, and the resulting mixture is placed in contact with biofilm. Alternatively, the solid peroxide can be dissolved in water and added to the biofilm. The manganese complex and the optional ligand of formula (I) or (II) (if not part of the manganese complex) can then be added to the mixture. Variations similar to those just discussed can be practiced when the aqueous mixture is a suspension.

[0054] Other variations are possible. For example, the manganese complex need not be added as such: manganese ions may be present or may be added to an aqueous mixture and a manganese complex formed in the aqueous mixture by adding an appropriate ligand of formula (I) or (II). Furthermore, and according to the second aspect of the invention, the biofilm may comprise sufficient quantities of manganese ions so that useful quantities of the manganese complexes described herein may be generated in the aqueous mixture by adding an appropriate amount of the Petition 870220076624, dated 08 / 25 / 2022, page 19 / 115 16 / 99 binders of formula (I) or (II) described herein.

[0055] According to the first aspect of the invention, the aqueous mixture that comes into contact with the biofilm comprises (i) a peroxide compound and (ii) a mononuclear manganese complex Mn(II), Mn(III) or Mn(IV), or a binuclear complex Mn(II)Mn(II), Mn(III)Mn(II), Mn(III)Mn(III), Mn(III)Mn(IV) or Mn(IV)Mn(IV), wherein the aqueous mixture comprises a ligand of formula (I) or (II).

[0056] It is not necessary that the ligand of formula (I) or (II) be part of the manganese complex, i.e., such ligand may be decomplexed in the aqueous mixture. In some embodiments, the manganese complex comprises the ligand, as in [MnIVMnIV(qO)3(1,4,7-trimethyl-1,4,7triazacyclononane)2][CH3COO]2. The aqueous mixture may comprise an excess of ligand so that the manganese complex comprises the ligand and the aqueous mixture comprises an additional uncomplexed ligand.

[0057] The aqueous mixtures used according to aspects of the invention and embodiments thereof comprise a ligand of formula (I) and / or (II), wherein R is independently selected from the group consisting of C1-C24 alkyl, CH2C6-C10 aryl, CH2CH2OH, CH2COOH and pyridin-2-ylmethyl.

[0058] If the biofilm comes into contact with the aqueous mixture (e.g., solution) disclosed in this document as a mixture, it may be a fluid paste, slurry, or suspension.

[0059] The term alkyl is well known in the art and defines univalent groups derived from alkanes by the removal of a hydrogen atom from any carbon atom, wherein the term alkane is intended to define branched or unbranched cyclic or acyclic hydrocarbons. If an alkyl is a C1C4 alkyl, it is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl.

[0060] The term aryl is also well known in the art and defines univalent groups derived from arenes by the removal of a hydrogen atom from a carbon atom of the ring, where the term arene is intended to define monocyclic and polycyclic aromatic hydrocarbons. The term “aromatic” Petition 870220076624, dated 08 / 25 / 2022, p. 20 / 115 17 / 99 defines a cyclically conjugated molecular entity with significantly greater stability (due to delocalization) than that of a hypothetical localized structure. Hückel's rule is frequently used in the technique to evaluate aromatic character; planar (or nearly planar) monocyclic systems of trigonally (or sometimes digontically) hybridized atoms containing (4n+2) π electrons (where n is a non-negative integer) will exhibit aromatic character. The rule is generally limited to an = 0 to 5.

[0061] R can be independently selected from the group consisting of C1-C24 alkyl, CH2C6-C10 aryl, CH2CH2OH and CH2COOH. Generally, the alkyl is a C1-C12 alkyl, so R is generally independently selected from the group consisting of C1-C12 alkyl, CH2C6-C10 aryl, CH2CH2OH and CH2COOH.

[0062] Typically, when R is an alkyl group, it is a C1-C6 alkyl group. Preferably, the alkyl group is a methyl group.

[0063] Generally, when R is CH2C6-C10aryl, this is a benzyl. Thus, in some embodiments, R is independently selected from the group consisting of C1-C6alkyl, benzyl, CH2CH2OH and CH2COOH.

[0064] R can be independently selected from C1-C6 alkyl or CH2C6-C10 aryl. In some embodiments, R is independently selected from C1-C6 alkyl or benzyl. Typically, R is independently selected from methyl or benzyl, preferably methyl.

[0065] Typically, each R is the same.

[0066] In formula (I) and formula (II), R1, R2, R3, and R4 are independently selected from H, C1-C4 alkyl, and C1-C4 hydroxyalkyl. The term “hydroxyalkyl” is used here to refer to univalent groups derived from alkyl groups by substitution of a hydrogen atom (-H) by a hydroxyl group (-OH). The C1-C4 hydroxyalkyl can be selected from the group consisting of hydroxymethyl, hydroxyethyl, n-hydroxypropyl, hydroxyisopropyl, n-hydroxybutyl, sec-hydroxybutyl, hydroxyisobutyl, and tert-hydroxybutyl. Preferably, the C1-C4 alkyl is methyl and the C1-C4 hydroxyalkyl is hydroxymethyl, thus R1, R2, R3, and R4 Petition 870220076624, dated 08 / 25 / 2022, page 21 / 115 18 / 99 are generally independently selected from H, methyl, and hydroxymethyl. Typically, R1, R2, R3, and R4 are independently H or methyl. Preferably, R1, R2, R3, and R4 are H.

[0067] When Q' is a propylene bridge in formula (II), this can be 1,3-propylene(-(CH2)3-) or 1,2-propylene(-CH2CH(CH3)-).

[0068] Generally, Q' is an ethylene bridge and the ligand of formula (II) is therefore represented by the following structure: RR ^-N\ ^N\ R2R1C ^CR3R4R4R3C''^ ^-cr1r2 R4R3C / Z\r.|R2 R2RiC / / \r3r4 R1R2 R3R4R3R4 R1R2 .

[0069] Generally, the ligand of formula (I) is 1,4,7-trimethyl-1,4,7-triazacyclononane (Me3-TACN) and the ligand of formula (II) is 1,2-bis(4,7-dimethyl-1,4,7-triazacyclonon-1-yl)ethane (Me4-DTNE). Thus, in some embodiments, the ligand is Me3-TACN or Me4-DTNE. In many embodiments, the ligand is Me3TACN.

[0070] According to the method of the first aspect of the invention, the manganese complex is a mononuclear complex of Mn(II), Mn(III) or Mn(IV), or a binuclear complex of Mn(II)Mn(II), Mn(III)Mn(II), Mn(III)Mn(III), Mn(III)Mn(IV) or Mn(IV)Mn(IV). Those skilled in the art are familiar with these complexes and salts of such complexes, which can form in aqueous mixture without isolation, or which can be well defined.

[0071] A well-defined complex is understood here (as the term is usually used in the art) to be a complex that has been isolated in such a way that it is susceptible to characterization (i.e., definition) and analysis (e.g., to determine its structure and degree of purity). In contrast, a complex that is not well-defined is one that is prepared without isolation from the medium (e.g., reaction medium) in which it is prepared. Petition 870220076624, dated 08 / 25 / 2022, page 22 / 115 19 / 99

[0072] Typically, the complex is a binuclear complex. However, the use of complex salts containing mononuclear manganese ions is also within the scope of the present invention. Examples of such complexes are described in patent applications EP 0549271 A1, EP 0549272 A1, EP 0544519 A2 and EP 0544440 A2 (all Unilever).

[0073] Mononuclear manganese complexes comprising a ligand of formula (I) comprise one ligand of formula (I) for each manganese ion to which the ligand of formula (I) coordinates. Binuclear manganese complexes comprising a ligand of formula (I) generally comprise two ligands of formula (I) for each two manganese ions, wherein each ligand of formula (I) is coordinated to one manganese ion. For example, when the ligand of formula (I) is Me3-TACN, and the manganese complex comprises the ligand, the manganese complex may be a mononuclear complex comprising one manganese ion and one Me3-TACN ligand or a binuclear complex comprising two manganese ions and two Me3-TACN ligands.

[0074] In contrast, binuclear manganese complexes comprising a ligand of formula (II) typically comprise one ligand of formula (II) for every two manganese ions and the ligand of formula (II) coordinates for each manganese ion in the complex. For example, when the ligand of formula (II) is Me4-DTNE and the manganese complex comprises the ligand, the manganese complex may be a binuclear complex comprising two manganese ions and one Me4-DTNE ligand.

[0075] As mentioned above, the aqueous mixture comprises excess ligand in certain embodiments, such that the manganese complex comprises the ligand and the aqueous mixture further comprises an uncomplexed ligand. As used in this document, excess ligand refers to a ratio of ligand to manganese ions resulting in an aqueous mixture comprising uncomplexed ligand. Consequently, excess ligand refers to a formula ratio (I) to manganese ions that is greater Petition 870220076624, dated 08 / 25 / 2022, page 23 / 115 20 / 99 that 1, and typically refers to a formula (II) ligand to manganese ion ratio that is greater than 0.5. When the aqueous mixture comprises excess ligand, it may comprise a mixture of uncomplexed ligand and poorly defined mononuclear and binuclear manganese complexes. For example, when the ligand is of formula (II), such as Me4-DTNE, the aqueous mixture may comprise a poorly defined mixture of binuclear complexes comprising two manganese ions and a Me4-DTNE ligand, mononuclear complexes comprising a manganese ion and a Me4-DTNE ligand (with one of the ligand's macrocyclic rings uncomplexed), and uncomplexed Me4-DTNE ligand.

[0076] In some embodiments, the manganese complex is well defined. The manganese complex may be well defined and the aqueous mixture further comprises a non-coordinated ligand.

[0077] Although the uncomplexed ligands of formula (I) or (II), which can be used according to the invention, on their own, i.e., in the absence of manganese ions, degrade the biofilm, the inventors have found that biofilm degradation is unexpectedly more effective when excess ligand (i.e., formula (I) or (II)) is present. Furthermore, as described above, a biofilm may contain manganese ions, which can bind to a ligand of formula (I) or (II), leading to biofilm degradation activity in the presence of a peroxide. Without being limited by theory, the presence of excess ligand can shift the equilibrium to favor ligand complexation on manganese.When practicing a method of the invention, if some of the ligands complexed with the manganese complexes become uncomplexed (and, for example, degrade or become unable to complex with manganese ions), then it can be replaced by the excess ligand in the aqueous mixture, thus regenerating manganese complexes comprising ligands of formula (I) or (II).

[0078] Typically, the molar ratio of a binder of formula (I) to manganese in the aqueous mixture of the first aspect of the invention is from about 100:1 to about Petition 870220076624, dated 08 / 25 / 2022, p. 24 / 115 21 / 99 of 0.1:1, more typically from about 10:1 to about 0.5:1, even more typically from about 5:1 to about 0.8:1 and most typically from about 2:1 to about 1.001:1. A molar ratio of about 1:1 refers to well-defined manganese complexes comprising a ligand of formula (I) with no uncomplexed ligand in the aqueous mixture, or to a mixture of manganese complex and a ligand of formula (I) in equimolar ratio.

[0079] Typically, the molar ratio of a ligand of formula (II) to manganese in the aqueous mixture of the first aspect of the invention is from about 50:1 to about 0.05:1, more typically from about 5:1 to about 0.1:1, even more typically from about 3:1 to about 0.2:1 and most typically from about 1:1 to about 0.5001:1. A molar ratio of 0.5:1 refers to well-defined manganese complexes comprising a ligand of formula (II) without any uncomplexed ligand in the aqueous mixture, or to a mixture of manganese complex and a ligand of formula (II) comprising two equivalents of manganese ions relative to the ligand of formula (II).

[0080] The manganese complexes used according to this invention may comprise coordination ligands in addition to the ligands of formula (I) or (II). When the manganese complex is a binuclear manganese complex, it may comprise one or more bridging ligands. These are typically selected independently from the group consisting of oxide, hydroxide, water, phenylboronate and R5COO-, wherein R5 is selected from the group consisting of hydrogen, C1-C12 alkyl and optionally phenyl substituted by C1C6 alkyl, whose ligands link the two manganese ions. Frequently, C1-C12 alkyl is a C1-C6 alkyl, particularly frequently a C1-C4 alkyl, and preferably methyl. Frequently, optionally C1-C6 alkyl substituted phenyl is a phenyl substituted by alkyl, optionally C1-C4, preferably a phenyl substituted by methyl.

[0081] The term substituted when used in this document is intended to refer to the substitution of a hydrogen atom in the referenced group with the referenced substituent(s). For example, optionally substituted phenyl Petition 870220076624, dated 08 / 25 / 2022, p. 25 / 115 22 / 99 for C1-C1-alkyl refers to a phenyl group in which one or more of the hydrogen atoms are optionally replaced by a C1-C1-alkyl group, typically by methyl, so that it gives, for example, benzyl.

[0082] R5 is typically selected from the group consisting of hydrogen (i.e., the bridging ligand is a formate), C1-C12 alkyl and phenyl optionally substituted with one or more methyl groups. Even more typically, R5 is selected from the group consisting of hydrogen, C1-C1 alkyl and phenyl. Even more typically, R5 is selected from the group consisting of hydrogen, C1-C4 alkyl and phenyl, wherein the C1-C4 alkyl is preferably methyl. Preferably, R5 is methyl or phenyl, with the greatest preference being methyl, i.e., if a carboxylate bridge is present, this is preferably either acetate or benzoate, with the greatest preference being acetate.

[0083] The one or more bridging ligands are, in particular embodiments, one or a combination selected from the group consisting of oxide, hydroxide, water, acetate and benzoate.

[0084] In some embodiments, the binuclear manganese complex comprises two or three bridging ligands, often three bridging ligands.

[0085] The manganese complex may be binuclear, that is, it comprises two manganese ions. Both ions may be Mn(II), Mn(III) or Mn(IV), one ion may be Mn(II) and the other Mn(III), or one ion may be Mn(III) and the other Mn(IV). In some cases, the binuclear manganese complex is an Mn(III)Mn(III), Mn(III)Mn(IV) or Mn(IV)Mn(IV) complex.

[0086] When the binuclear manganese complex is an Mn(III)Mn(III) complex, it typically comprises one bridging oxide ligand and two bridging carboxylate ligands (R5COO-). Frequently, the carboxylate ligands are acetate ligands. When the Mn(III)Mn(III) complex comprises two Me3-TACN ligands, it is typically [MnIIIMnIII(pO)(pCH3COO)2(Me3-TACN)2]2+.

[0087] When the binuclear complex is an Mn(III)Mn(IV) complex, it preferably comprises three bridging ligands; typically one or two Petition 870220076624, dated 08 / 25 / 2022, page 26 / 115 23 / 99 oxide-bridge ligands and two or one acetate-bridge ligands. When the Mn(III)Mn(IV) complex comprises two ligands of formula (I) (for example, when two Me3-TACN ligands are chelating Mn ions), it typically comprises one oxide-bridge ligand and two acetate-bridge ligands. Thus, the binuclear complex may be [MnIIIMnIV(pO)(p-CH3COO)2(Me3TACN)2]3+. In contrast, when the Mn(III)Mn(IV) complex comprises a ligand of formula (II) (for example, when a Me4-DTNE is chelating both Mn ions in the complex), it typically comprises two oxide-bridge ligands and one acetate-bridge ligand. Thus, the binuclear complex is typically [MnIIIMnIV(pO)2(p-CH3COO)(Me4-DTNE)]2+.

[0088] When the binuclear complex is an Mn(IV)Mn(IV) complex, it typically comprises two or three oxide-bridge ligands and none or one acetate-bridge ligand. When the Mn(IV)Mn(IV) complex comprises two ligands of formula (I) (for example, when two Me3-TACN ligands are chelating Mn ions), it typically comprises three oxide-bridge ligands. Thus, the binuclear complex may be [MnIVMnIV(pO)3(Me3-TACN)2]2+. In contrast, when the Mn(IV)Mn(IV) complex comprises one ligand of formula (II) (for example, when a Me4-DTNE is chelating both Mn ions in the complex), it typically comprises two oxide-bridge ligands and one acetate-bridge ligand. Thus, the binuclear complex is typically [MnIVMnIV(pO)2(p-CH3COO)(Me4DTNE)]3+.

[0089] In some embodiments, the manganese complex is selected from any group that consists of [MnIIIMnIII(pO)(p-R5COO)2(Me3TACN)2]2+, [MnIIIMnIV(pO)(p-R5COO)2(Me3-TACN)2]3+, [MnIVMnIV(pO)3(Me3TACN)2]2+, [MnIIIMnIV(pO)2(p-R5COO)(Me4-DTNE)]2+e [MnIVMnIV(pO)2(pR5COO)(Me4-DTNE)]3+, em que R5é conforme descrito acima. Preferably, R5 is methyl.

[0090] Typically, the manganese complex is selected from any group that consists of [MnIIIMnIII(pO)(p-CH3COO)2(Me3-TACN)2]2+, Petition 870220076624, de 25 / 08 / 2022, pág. 27 / 115 24 / 99 [Mmmmm(m-0)(m-CH3000)2(Mg3-TAON)2]3+, [Mmmmm(m-0)3(Mg3-TAON)2]2+, [MniiiMn(μ-O)2(μ-OH3000)(Mn4-CTNE)]2+θ [MniiiMn(μ-O)2(μ-OH3000)(Mn4DTNE)]3+.

[0091] In general, the structure of the compound is [MnMn(μ-0)3(Mn3-TAON)2]2+ or [MnMn(μ-0)2(μ-K5000)(Mn4^TNE)]2+, where R5 is the upper limit of the above. In general, the structure of the compound is the upper limit of the above, R5 is the lower limit.

[0092] As mentioned above, the manganese oxide of the invention is not a pure manganese oxide. For this purpose, two or three ligands of the manganese oxide are used, which, upon exposure to air, form the pure manganese oxide species Mn(III)Mn(III), Mn(III)Mn(IV) and Mn(IV)Mn(IV). These various complexes of Mn(II) are formed in the aqueous mixture, for example, when the aqueous mixture contains one or more manganese salts, for example, Mn(II)(acetate)2, and the ligand is a compound of formula (i) or (ii), for example, Mn(II)(acetate).

[0093] Many times, mononuclear or beta-nuclear manganese is positively charged. In other words, the positive charge is balanced by one or more non-chromatin counter-anions. A ίόθ^^όθ όο^) ^η^-άηιοη^) não é uma características θssθncial όa invθnção, θmbora, para πθΙΜογ solubiliόaόθ θπ πθιο aquoso, contraíons muifo granόθs, αοπο tθtrafθnilborato, sθjam aliaόos (θmbora não nθcθssariamθntθ) θvitaόos.

[0094] Frθquθntθmθntθ, non-corresponding counterions are isolated from any group that consists of θπ ΟΙ-, Br-, ι-, Ν03-, ΟΙ04-, PF6-, S042-, R6S03-θ R5O00-, θπ quθ R5é ^πο όθwritten above θπ relation to the ligands θπ ρο^θ όθ carboxylate; θ R6is optionalmθntθ try Ο1-Ο6alkyl substitute, Oi^a^uite (ροΓ θχθπρΙο, πθ^^) or OF3. When R6 is a substitute Hi^a^uite attempt, the attempt is only a substitute for one or more ram vθzθs of Hi^a^uite. Τίρ^πθ^θ, when R6 is a substitute attempt, it is Petition 870220076624, dated 08 / 25 / 2022, p. 28 / 115 25 / 99 is a substituted C1-C4 alkyl phenyl group, in which C1-C4 alkyl is preferably methyl. R6 may be phenyl optionally substituted by one or more methyl groups. Often, the phenyl group is substituted by a methyl group, typically in the para position.

[0095] In some embodiments, the non-coordinating counterions are selected from the group consisting of SO42-, R5COO-, Cl-, NO3-, R6SO3-, and PF6-. Frequently, the non-coordinating counterions are selected from the group consisting of SO42-, CH3COO-, Cl-, NO3-, CH3C6H4SO3- (tosylate), and PF6-.

[0096] Salts comprising manganese complexes and having significant water solubility, such as at least 30 g / l at 20 °C, e.g., at least 50 g / l at 20 °C or at least 70 g / l at 20 °C, are described in document WO 2006 / 125517 A1 (Unilever PLC). The use of such highly water-soluble salts, i.e., salts with solubilities of at least 30 g / l at 20 °C, e.g. at least 50 g / L at 20 °C or at least 70 g / L at 20 °C, such as and typically (but not necessarily) those described in document WO 2006 / 125517 A1 (Unilever PLC), for example, those comprising small counterions such as chloride, nitrate, sulfate and acetate, may be advantageous, since their high solubilities in water mean, for example, that higher concentrations of the salts can be used in the aqueous mixtures of the invention than when using salts that are poorly soluble in water, such as those comprising the PF6- ion.For example, the water solubility of [MnIVMnIV(pO)3(Me3-TACN)2][PF6]2 is only 10.8 g / L at 20 °C.

[0097] Furthermore, poorly water-soluble salts, such as those comprising PF6-, are typically formed by introducing the anion (PF6-) as a potassium salt after the formation of the manganese complex, leading to the precipitation of the salt comprising the manganese complex and the poorly water-soluble counterion (PF6-). The precipitate is typically redissolved, for example, in water, before addition to the aqueous mixture disclosed according to the first aspect of the invention. Such additional steps introduce complexity and cost, as well as Petition 870220076624, dated 08 / 25 / 2022, page 29 / 115 26 / 99 often necessitates the use of relatively large volumes of water or other solvent, since its solubility (in water) is quite low.

[0098] Thus, it is preferable that the non-coordination counterions be selected from the group consisting of Cl-, NO3-, SO42- and acetate. However, when the non-coordination counterion is PF6, the manganese complex will typically be [MnIVMnIV(pO)3(Me3-TACN)2]2+, i.e., the manganese salt will typically be [MnIVMnIV(pO)3(Me3-TACN)2][PF6]2.

[0099] In some embodiments, the manganese complex is part of a salt selected from any of the group consisting of [MnIIIMnIII(pO)(pR5COO)2(Me3-TACN)2][CH3COO]2, [MnIIIMnIV(pO)(p-R5COO)2(Me3TACN)2][CH3COO]3, [MnIVMnIV(pO)3(Me3-TACN)2][CH3COO]2, [MnIIIMnIII(gO)(p-R5COO)2(Me3-TACN)2][SO4], [MnIIIMnIV(pO)(p-R5COO)2(Me3TACN)2]2[SO4]3, [MnIVMnIV(pO)3(Me3-TACN)2][SO4], [MnIIIMnIII(pO)(pR5COO)2(Me3-TACN)2][NO3]2, [MnIIIMnIV(pO)(p-R5COO)2(Me3-TACN)2][NO3]3, [MnIVMnIV(pO)3(Me3-TACN)2][NO3]2, [MnIVMnIV(pO)3(Me3-TACN)2][PF6]2, [MnIIIMnIV(pO)2(p-R5COO)(Me4-DTNE)][Cl]2 and [MnIVMnIV(pO)2(p-R5COO)(Me4DTNE)][Cl]3, where R5 is as described above. Preferably, R5 is methyl.

[0100] Preferably, the manganese complex is part of a salt selected from any of the group consisting of [MnIVMnIV(pO)3(Me3TACN)2][CH3COO]2, [MnIVMnIV(pO)3(Me3-TACN)2][SO4], [MnIVMnIV(pO)3(Me3TACN)2][NO3]2 or [MnIIIMnIV(pO)2(p-R5COO)(Me4-DTNE)][Cl]2, where R5 is as described above. Preferably, R5 is methyl.

[0101] It should be understood that the salt comprising the manganese complex may (in its solid form) contain additional water molecules, known in the art as hydrates. For example, crystalline [MnIVMnIV(pO)3(Me3TACN)2][PF6]2 typically comprises a water molecule within its crystal lattice. The molecular formula of the hydrate is [MnIVMnIV(pO)3(Me3TACN)2][PF6]2.H2O.

[0102] A solid manganese salt (comprising the manganese complex) can be synthesized in such a way that it is isolated in combination with salts Petition 870220076624, dated 08 / 25 / 2022, p. 30 / 115 27 / 99 additional. During the synthesis of the manganese salt, an excess of the reagent used to provide the desired counterion may have been used and may not have been separated from the resulting solid comprising the manganese salt. For example, potassium hexafluorophosphate may be present in solid [MnIVMnIV(pO)3(Me3-TACN)2][PF6]2 and sodium chloride may be present in [MnIIIMnIV(p-O)2(p-R5COO)(Me4-DTNE)][Cl]2 as exemplified in Example 2 of document WO 2013 / 033864 A1 (Kemp, RW et al).

[0103] Alternatively, and according to the second aspect, it is contemplated that the aqueous mixture in contact with the biofilm comprises a peroxide compound and a ligand of formula (I) or (II) without requiring the presence of manganese ions. The solvent used to make the aqueous mixture may contain manganese ions as an impurity, which may bind to the ligand of formula (I) or (II) to form mononuclear or binuclear (not well defined) manganese complexes. Similar observations have been made regarding the bleaching of tea stains, where notable bleaching effects were observed when using a salt of the protonated Me3-TACN ligand in combination with hydrogen peroxide (G. Reinhardt, J. Molecular Catalysis, 2006, 251, 177-184).Furthermore, biofilms may also contain manganese ions which, after the addition of a ligand of formula (I) or (II), will form Mn(II), Mn(III), Mn(IV) or not well-defined binuclear mononuclear Mn(II)Mn(II), Mn(III)Mn(II), Mn(III)Mn(III), Mn(III)Mn(IV), or Mn(IV)Mn(IV) complexes.

[0104] If, according to the second aspect of the invention, the aqueous mixture in contact with the biofilm comprises a peroxide and binder compound of formula (I) or (II) without requiring the presence of manganese ions, the concentration of manganese complex formed in the aqueous mixture is typically from about 0.0001 to about 300 qM, more typically from about 0.001 to about 200 qM, even more typically from about 0.01 to about 100 qM, and even more typically from about 0.1 to about 50 qM. Frequently, the concentration of manganese complex is from about 0.01 to about 30 qM and even more frequently from about 0.05 to about 20 qM. Petition 870220076624, dated 08 / 25 / 2022, p. 31 / 115 28 / 99

[0105] Upon contacting the aqueous mixtures disclosed in this document with biofilm, the biofilm is degraded. Surprisingly, the extent of the degree of adaptation was found by the inventors to be greater when, prior to contact, a manganese compound is brought into contact with a reducing agent to provide the manganese complex. It should be understood that a manganese compound refers to any compound containing manganese ions which, upon contact with a reducing agent, forms the manganese complex referred to in the first aspect of the invention. Without being limited by theory, the addition of a reducing agent can lower the oxidation state of the manganese ion or ions present in the manganese compound. Thus, the manganese ions within the manganese compound are of a higher oxidation state than the manganese ions within the corresponding manganese complex, which forms upon contacting the manganese compound with a reducing agent.In view of this, the reducing agent is generally brought into contact with manganese compounds comprising manganese ions with an oxidation state of 3 or higher. Typically, the reducing agent is brought into contact with a mononuclear Mn(III) or Mn(IV) compound, or a binuclear Mn(III)Mn(III), Mn(III)Mn(IV), or Mn(IV)Mn(IV) compound. In some examples, the manganese compound is a binuclear compound of Mn(III)Mn(III), Mn(III)Mn(IV), or Mn(IV)Mn(IV).

[0106] As an example, the addition of a molar equivalent of a two-electron reducing compound, such as ascorbic acid, to a binuclear Mn(IV)Mn(IV) compound can lead to the reduction of both Mn(IV) ions to produce a binuclear Mn(III)Mn(III) complex. However, it is not necessarily the case that a well-defined binuclear Mn(III)Mn(III) complex is formed when a molar equivalent of a two-electron reducing agent, such as ascorbic acid, is added to a well-defined binuclear Mn(IV) compound, such as a manganese compound comprising [MnIVMnIV(pO)3(Me3TACN)2]2+. Instead, different manganese complexes may be present in the resulting mixture, for example, a mixture of binuclear Petition 870220076624, dated 08 / 25 / 2022, page 32 / 115 29 / 99 Mn(II)Mn(II), Mn(III)Mn(II), Mn(III)Mn(III), Mn(III) Complexes of Mn(IV) or Mn(IV)Mn(IV) can form (where the average oxidation state is Mn(III)Mn(III)). It may also be possible that ligand dissociation occurs leading to the formation of mononuclear manganese complexes, i.e., Mn(II), Mn(III) and / or Mn(IV) complexes, see, for example, BC Gilberto, et al. Org. Biomol. Chem., 2004, 2, 1176-1180, or of course, a less than complete reduction occurs (i.e., less than that calculated stoichiometrically).

[0107] When the manganese complex is provided by contacting a manganese compound with a reducing agent, the manganese complex is normally provided in a solution (typically an aqueous solution). Such a solution may be formed by any suitable method. For example, the manganese compound may be obtained or prepared as a solution or suspension and may be added as a solution or suspension to an optionally buffered solvent (typically water) before, after, or with a reducing agent. Alternatively, if the manganese compound is obtained or prepared as a solid, it may be added to the optionally buffered solvent (typically water) before contact with the reducing agent. When the reducing agent is a solid (such as ascorbic acid), it may be mixed with the manganese compound and the resulting mixture may then be added to the solvent.Alternatively, the reducing agent may be supplied in a solution or suspension (typically an aqueous solution or suspension).

[0108] The manganese compound may be selected from the group consisting of binuclear complexes of Mn(III)Mn(III), Mn(III)Mn(IV) and Mn(IV)Mn(IV) and, upon contact with the reducing agent, may provide any one or a combination of manganese complexes selected from the group consisting of mononuclear complexes of Mn(II), Mn(III) and Mn(IV), and binuclear complexes of Mn(II)Mn(II), Mn(III)Mn(II), Mn(III)Mn(III) and Mn(III)Mn(IV).

[0109] The reducing agent can be any reducing agent suitable for reducing the oxidation state of the manganese compound. Reducing agents Petition 870220076624, dated 08 / 25 / 2022, page 33 / 115 Suitable 30 / 99 compounds include ascorbic acid and ester derivatives such as ascorbyl palmitate or ascorbyl stearate, optionally C1-C4 alkyl- or allyl-substituted catechol, optionally C1-C4 alkyl-substituted hydroquinone, pyrogallol, caffeic acid, optionally C1-C4 alkyl-substituted maltol, n-propyl gallate, and alkali metal sulfites, alkali metal bisulfites, and alkali metal thiosulfates.

[0110] The alkali metal in sulfites, bisulfites and thiosulfates is usually sodium or potassium, typically sodium.

[0111] The reducing agent may be any one selected from the group consisting of ascorbic acid, ascorbyl palmitate, ascorbyl stearate, catechol, hydroquinone, pyrogallol, 4-tert-butylcatechol, 4-allylcatechol, tert-butylhydroquinone, 2,5-di-tert-butylhydroquinone, n-propyl gallate, caffeic acid, maltol, ethylmaltol, sodium sulfite, sodium bisulfite and sodium thiosulfate.

[0112] The reducing agent is generally any one selected from the group consisting of ascorbic acid, ascorbyl palmitate, ascorbyl stearate, catechol, hydroquinone, pyrogallol, sodium sulfite, sodium bisulfite and sodium thiosulfate.

[0113] In some embodiments, the reducing agent is selected from the group consisting of ascorbic acid, catechol, hydroquinone, pyrogallol, and sodium sulfite. According to particular embodiments, the reducing agent is ascorbic acid.

[0114] Generally, when the reducing agent is ascorbic acid, the manganese compound comprises a hexafluorophosphate counterion, for example, the manganese compound is [MnIVMnIV(pO)3(Me3-TACN)2][PF6]2.

[0115] The molar ratio of the manganese compound comprising, for example, a binuclear complex Mn(III)Mn(III), Mn(III)Mn(IV) or Mn(IV)Mn(IV), to the reducing agent is typically from about 0.1:1 to about 10:1, more typically from about 0.2:1 to about 3:1, and most typically from about 0.5:1 to about 1:1, i.e., about one or two molar equivalents of a reducing agent is added to the manganese compound. A large excess of reducing agent relative to the manganese compound may not be desirable, as the Petition 870220076624, dated 08 / 25 / 2022, page 34 / 115 31 / 99 Excess reducing agent may react with the peroxide compound present in the aqueous mixture. On the other hand, a large excess of manganese compound may not be desirable, as most of the manganese compound may not react with the reducing agent, thus remaining in a less active form.

[0116] In certain embodiments of the first aspect of the invention, the manganese compound comprises one or more of the ligands of formula (I) or one of the ligands of formula (II); two or three, preferably three, of the bridging ligands described herein; and / or one or more of the non-coordinating counterions described in this document. It is not necessary that the ligand of formula (I) or (II) be part of the manganese compound. Such a ligand may be added separately to the aqueous mixture and may come into contact with the transition metal compound in the formation of the transition metal complex, or may remain uncomplexed in the aqueous mixture. For the avoidance of doubt, the embodiments of the first aspect of the invention that apply to the manganese complex apply mutatis mutandis to the manganese compound. For example, the manganese compound may comprise a ligand of formula (I) or (II), which is preferably Me3-TACN or Me4-DTNE.The manganese compound may comprise one or more non-coordinating counterions selected from the group consisting of SO42-, R5COO-, Cl-, NO3-, R6SO3- and PF6-, wherein R5 and R6 are as defined herein. Preferably, one or more non-coordinating counterions are selected from the group consisting of acetate, chloride, sulfate, nitrate and hexafluorophosphate, i.e., acetate, chloride, sulfate and nitrate.

[0117] In another example, the manganese compound may be binuclear and, when binuclear, may comprise two or three independently selected bridging ligands consisting of oxide, hydroxide, water, phenylboronate and R5COO- where R5 is as defined in this document (preferably methyl).

[0118] In yet another example, the manganese compound may comprise any of the group consisting of [MnIIIMnIII(gO)(g-R5COO)2(MeTACN)2]2+, [MnIIIMnIV(gO)(g-R5COO)2(Me3-TACN)2]3+, [MnIVMnIV(gO)3(Me3 Petition 870220076624, dated 08 / 25 / 2022, page 35 / 115 32 / 99 TACN)2]2+, [MnIIIMnIV(pO)2(p-R5COO)(Me4-DTNE)]2+and [ΜηινΜηιν(μ-0)2(μR5COO)(Me4-DTNE)]3+, in which R5é is as defined herein (preferably methylated).

[0119] The manganese compound can be any one of the group consisting of [MnIIIMnIII^-O)^-R5COO)2(Me3-TACN)2][CH3COO]2, [MnIIIMnIV^-O)^R5COO)2(Me3-TACN)2][CH3COO]3,3 [MnIVMnI%-O)3(Me3-TACN)2][CH3COO]2, [MnIIIMnIII^-O)^-R5COO)2(Me3-TACN)2][SO4], [Mn^Mn^^fa-R^OO^MeeTACN)2]2[SO4]3, [MnIVMnI%-O)3(Me3-TACN)2][SO4], [Mn^MnIIVO)^R5COO)2(Me3-TACN)2][NO3]2, [MnIIIMnIV^-O)^-R5COO)2(Me3-TACN)2][NO3]3, [MnIVMnI%-O)3(Me3-TACN)2][NO3]2, [MnIVMnI%-O)3(Me3-TACN)2][NO3]2, [MnIVMnI%-O)2(Me3-TACN)2][NO3]2 [MnIIIMnI%-O)2^-R5COO)(Me4-DTNE)][Cl]2 and [MnIVMnI%-O)2^-R5COO)(Me4DTNE)][Cl]3, wherein R5E as herein defined (preferably methylated).

[0120] In another example, the manganese compound may comprise [MnIVMnI%-O)3(Me3-TACN)2]2+ or [MnIIIMnI%-O)2^-R5COO)(Me4-DTNE)]2+, wherein R5 is as defined herein (preferably methyl).

[0121] In yet another example, the manganese compound can be [MnIVMnI%-O)3(Me3-TACN)2][CH3COO]2, [MnIVMnI%-O)3(Me3-TACN)2][SO4], [MnIVMnI%-O)3(Me3-TACN)2][NO3]2 or [MnIIIMnI%-O)2^-CH3COO)(Me4DTNE)][Cl]2.

[0122] The aqueous mixture, according to the first and second aspects of the invention, comprises a peroxide compound. As used in this document, a peroxide compound is a compound of structure ROOR', where R and R' can independently be hydrogen or organyl.

[0123] Organelle as used in this document and understood in the art to refer to an organic substituent with a free valence on a carbon atom. Similarly, organylene refers to an organic group with two free valences, which may be on the same or different carbon atoms derived from the removal of two hydrogen atoms from an organic compound. Thus, for example, an organelle may be any one selected from the group consisting of -C(O)R'', C1-C12 alkyl and phenyl-C1-C4 alkyl, where R'' Petition 870220076624, dated 08 / 25 / 2022, p. 36 / 115 33 / 99 is an alkylene or substituted alkylene group. R and R' may be the same or different organyl groups. Where R is hydrogen and R' is -C(O)R'', the peroxide compound is a peroxyacid. Where R is hydrogen and R' is alkyl, the peroxide is an alkyl hydroperoxide. Where R is hydrogen and R' is phenylalkyl, it is a phenylalkyl hydroperoxide. Where R and R' are alkyl, the peroxide is a ketone peroxide. Peroxy acid, alkyl hydroperoxide, phenylalkyl hydroperoxide, and ketone peroxide are further defined in this document.

[0124] The peroxide compound may be any one or a combination of the group consisting of hydrogen peroxide, a peroxyacid, an alkyl hydroperoxide, a phenyl alkyl hydroperoxide, and a ketone peroxide.

[0125] Frequently, peroxide is any one or a combination of hydrogen peroxide, peroxyacid, alkyl hydroperoxide, and phenylalkyl hydroperoxide. Most often, peroxide is a combination of hydrogen peroxide and a peroxyacid. One or more different peroxide compounds may be used in combination.

[0126] The term peroxyacid is used in this document to refer to acids, such as carboxylic acids, in which at least one acidic -OH group has been replaced by an -OOH group. Typical mono- or diperoxyacids are of the general formula HOO(CO)R''Y, where R is an alkylene or substituted alkylene group containing from 1 to about 20 carbon atoms, optionally having an internal amide linkage or a phenylene or substituted C1-C12 alkyl phenylene group; and Y is hydrogen, halogen, C1-C12 alkyl, C6-C10 aryl (preferably phenyl), imide, a COOH or (C=O)OOH group or a quaternary ammonium group.

[0127] By imide is meant a diacyl derivative of ammonia or primary amines, i.e., comprising the structure R'-C(O)NRC(O)-R', wherein the R' groups are independently selected organyl groups, or more typically, are joined together by an organylene group connecting carbonyl moieties, so as to provide a cyclic imide; and R- represents the remainder of the peroxyacid, i.e., HOO(CO)R''-. Typically, the imide is cyclic, for example one of the group Petition 870220076624, dated 08 / 25 / 2022, page 37 / 115 34 / 99, which consists of phthalimide, maleimide, succinimide, and glutarimide. Preferably, the imide is phthalimide.

[0128] In some embodiments, R'' is an optionally substituted C1-C12 alkylene or phenylene; and Y is -H, halo, C1-C12 alkyl, phenyl, phthalimide, -COOH or -(C=O)OOH or a quaternary ammonium. Typically, R is an optionally substituted C1-C4 alkylene or phenylene; and Y is -H, halo or C1-C4 alkyl, wherein C1-C4 alkylene is selected from any of the group consisting of methylene, ethylene, n-propylene, isopropylene, n-butylene, sec-butylene, isobutylene and tert-butylene, preferably methylene, and C1-C4 alkyl is preferably methyl. More typically, R is an optionally substituted C1-C4 alkylene or phenylene; and Y is -H, or halo. Usually, halo is chlorine or fluorine.

[0129] Examples of mono-peroxyacids include peracetic acid, trifluoroperacetic acid, phthaloyl peroxide, a peroxybenzoic acid such as meta-chloroperbenzoic acid, peroxylauric acid, N,N-phthaloylaminoperoxycaproic acid and 6-octylamino-6-oxo-peroxyhexanoic acid.Typical diperoxyacids include, for example, 1,12-diperoxydecanoic acid and 1,9-diperoxyazeleic acid.

[0130] Typically, a peroxyacid is any one or a selected combination of peracetic acid, trifluoroacetic peracid, and meta-chloroperbenzoic acid.

[0131] The term alkyl hydroperoxide refers to monosubstituted products of hydrogen peroxide, in which an -H is replaced by an alkyl group. Alkyl can be a C1-C12 alkyl, and is often a C1-C4 alkyl, preferably tert-butyl.

[0132] The term phenylalkylhydroperoxide refers to monosubstitution products of hydrogen peroxide, in which an -H is replaced by a phenylalkyl group. The phenylalkyl may be a C1-C4 phenylalkyl selected from the group consisting of phenylmethyl, phenylethyl, n-phenylpropyl, phenylisopropyl, n-phenylbutyl, sec-phenylbutyl, phenylisobutyl and tert-phenylbutyl, frequently phenylisopropyl. Petition 870220076624, dated 08 / 25 / 2022, page 38 / 115 35 / 99

[0133] The term ketone peroxide refers to a peroxide compound that forms upon contact with a ketone and hydrogen peroxide. Such peroxide compounds are often not well-defined and can form in aqueous mixtures upon contact with a ketone and hydrogen peroxide. Suitable ketones include acetone, methyl ethyl ketone (butanone), methyl propyl ketone, methyl isopropyl ketone, ethyl propyl ketone, methyl phenyl ketone, and diphenyl ketone. Ketone peroxide is typically methyl ethyl ketone peroxide or acetone peroxide.

[0134] The peroxide compound of the first and second aspects of the invention is frequently obtained or prepared as an aqueous solution (optionally diluted, for example, with water or alkaline buffers), such as an aqueous solution comprising any one or a combination selected from the group consisting of hydrogen peroxide, peroxyacid, alkyl hydroperoxide and phenyl alkyl hydroperoxide. Handling liquid peroxide compounds is generally easier.

[0135] The peroxide compound of the first and second aspects can be formed in the aqueous mixture from a suitable precursor. Suitable precursors of peroxide compounds are known in the art and the person skilled in the art is able to identify appropriate precursors for use according to the first and second aspects of the invention. When the peroxide compound is hydrogen peroxide, it can be formed in the aqueous mixture from precursors including alkali metal peroxides, organic peroxides such as urea hydrogen peroxide, and inorganic persalts such as alkali metal perborates (e.g., sodium perborate), percarbonates, perphosphates, persilicates, and persulfates such as potassium monopersulfate. Often, the persalt is optionally hydrated sodium perborate (e.g., sodium perborate monohydrate and sodium perborate tetrahydrate) or sodium percarbonate.Sodium percarbonate degrades into hydrogen peroxide and sodium carbonate. It is generally considered more environmentally friendly than other sources of hydrogen peroxide and, consequently, is more economical. Petition 870220076624, dated 08 / 25 / 2022, page 39 / 115 36 / 99 is widely used as a solid source of hydrogen peroxide.

[0136] Other suitable sources of hydrogen peroxide include enzymatic systems which, together with a suitable substrate, produce hydrogen peroxide. An example of this is a C1-C4 alcohol oxidase enzyme and a C1-C4 alcohol, for example, a combination of methanol oxidase and ethanol. Such combinations are described in document WO 95 / 07972 A1 (Unilever NV and Unilever plc).

[0137] When the peroxide compound is a peroxyacid, it can form in the aqueous mixture from a so-called peroxy precursor. The peroxy precursor can react with hydrogen peroxide to generate peroxy acid. Peroxy precursors are well known to those skilled in the art and are described in documents GB 836988 A (Unilever Ltd), GB 864798 A (Unilever Ltd), GB 907356 A (Konink ind Mij Voorheen Noury), GB 1003310 A (Unilever Ltd) and GB 1519351 A (Unilever Ltd); EP 0185522 A2 (Clorox Co), EP 0174132 A2 (Proctor & Gamble), EP 0120591 A1 (Proctor & Gamble); and US 1246339 A (Smit Isaac J), US 3332882 A (FMC Corp), US 4128494 A (Ugine Kuhlmann), US 4412934 A (Proctor & Gamble) and US 4675393 A (Lever Brothers Ltd).

[0138] Suitable peroxy precursors include quaternary ammonium-substituted cationic peroxy acid bleaching precursors described in US 4751015 A and US 4397757 A (both Lever Brothers Ltd); and in EP 0284292 A (Kao Corp) and EP 0331229 A (Unilever NV). Examples of these include 2-(N,N,N-trimethylammonium)ethyl sodium-4-sulfophenyl carbonate (SPCC) chloride and N,N,N-trimethylammonium tolyloxybenzene sulfonate.

[0139] Another class of peroxy precursors is formed by the cationic nitriles described in EP 0303520 A (Kao Corp), EP 0458396 A (Unilever NV) and EP 0464880 A (Unilever NV). Other classes of bleaching precursors for use with the present invention are described in WO 00 / 15750 A1 (Proctor & Gamble), for example, 6-(nonanamidocaproyl)oxybenzenesulfonate.

[0140] Typically, the peroxy precursor is selected from the group consisting of an ester, including a sulfophenyl alkanoate and a sulfophenyl phenylalkanoate; Petition 870220076624, dated 08 / 25 / 2022, page 40 / 115 37 / 99 an acyl-amide; and a quaternary ammonium-substituted peroxy precursor, including a cationic nitrile. Examples of typical peroxy precursors (sometimes referred to as peroxy acid bleaching activators) are sodium 4-benzoyloxybenzene sulfonate (SBOBS); N,N,N',N'tetraacetylethylenediamine (TAED); sodium 1-methyl-2-benzoyloxybenzene-4-sulfonate; sodium 4-methyl-3-benzoyloxybenzoate; trimethylammonium tolyloxybenzene sulfonate; SPCC; sodium nonanoyloxybenzene sulfonate (SNOBS); sodium 3,5,5-trimethylhexanoyloxybenzene sulfonate; and the substituted cationic nitriles. Frequently, the peroxy precursor is TAED or nonanoyloxybenzene sulfonate (NOBS) salts, e.g., SNOBS.

[0141] When more than one peroxide compound is used, the combination may be selected from the group consisting of hydrogen peroxide, peroxyacid, C1-C12 alkyl hydroperoxide, and phenyl alkyl hydroperoxide. When there is more than one peroxide compound in the aqueous mixture, these are typically selected from hydrogen peroxide and any peroxide compound selected from the group consisting of peroxyacid, C1-C12 alkyl hydroperoxide, and phenyl alkyl hydroperoxide. Generally, hydrogen peroxide is the most effective peroxide compound for a method of the invention. However, if catalase enzymes are present in the aqueous mixture (e.g., produced by microorganisms within the biofilm), or transition metal ions that preferentially react with hydrogen peroxide, then the amount of active hydrogen peroxide in the aqueous mixture decreases.The manganese complex of the invention can instead react with the peroxide compound selected from the group consisting of peroxyacid, C1-C12 alkyl hydroperoxide, and phenyl alkyl hydroperoxide. Thus, the use of a peroxide compound other than hydrogen peroxide may be advantageous in a method of the invention. For example, the antimicrobial properties of peroxyacids are well known, and when degrading biofilms, antimicrobial activity is likely to be desirable (for antimicrobial properties of peroxyacids, see Katara, G. et al., J. Patient Saf. Infect. Control, 2016, 4(1), 17-21; Shen, X. et al., Front. Petition 870220076624, dated 08 / 25 / 2022, page 41 / 115 38 / 99 Microbiol., 2019, 10, 1196; Antonelli, M. et al., Water Sci. Technol., 2013, 68(12), 2638-2644; and WO 2017 / 181005 A1 (Ecolab USA Inc.)). Frequently, a peroxyacid is used in a method of the invention in combination with hydrogen peroxide, often with a higher molar ratio of hydrogen peroxide used relative to the peroxide.

[0142] In some embodiments, a mixture of C1-12 alkyl hydroperoxides (preferably tert-butyl hydroperoxide) and hydrogen peroxide is used. Generally, the molar ratio between C1-12 alkyl hydroperoxide and hydrogen peroxide is about 10:1 to about 1:10.

[0143] Alternatively, a mixture of peroxyacids (typically selected from the group consisting of peracetic acid, metachloroperbenzoic acid, trifluoroacetic peracid and phthaloyl peroxide, preferably peracetic acid) and hydrogen peroxide is used. Frequently, the molar ratio of peroxyacid to hydrogen peroxide is from about 10:1 to about 1:100, preferably from about 5:1 to about 1:10.

[0144] Often, a mixture of peracetic acid and hydrogen peroxide is used. Typically, the molar ratio of peracetic acid to hydrogen peroxide is about 10:1 to about 1:100, more typically about 3:1 to about 1:30, and even more typically about 1:1 to about 1:10.

[0145] The concentration of the peroxide compound can vary. For the avoidance of doubt, the concentration of the peroxide compound refers to the total concentration of all peroxide compounds in the aqueous mixture, including hydrogen peroxide, peroxyacid, alkyl hydroperoxide, phenylalkyl hydroperoxide, and ketone hydroxide. Typically, the concentration of the peroxide compound is about 0.01 to about 500 mM, more typically about 0.1 to about 100 mM, and most typically about 0.3 to about 30 mM.

[0146] The inventors discovered that the biofilm degrades upon contact with the aqueous solution of the first aspect of the invention comprising the peroxide compound, manganese complex and binder of formula (I) or (II). If the binder or peroxide compound is removed or degraded, there will no longer be any biofilm. Petition 870220076624, dated 08 / 25 / 2022, p. 42 / 115 39 / 99 biofilm degradation.

[0147] The method of the first and second aspects of the invention can be carried out at a variety of temperatures and pH ranges. Degradation of the manganese complex can occur when the pH of the aqueous mixture is outside a suitable range. When the aqueous mixture has a high pH value, insoluble manganese hydroxides / oxides can form, for example, at a pH >12 or >13. Furthermore, degradation of the peroxide compound by the manganese complex can occur at high pH values, for example, at a pH > 10.5. Such degradation of the peroxide compound can inhibit biofilm degradation. However, this can be easily avoided, for example, by increasing the concentration of the peroxide compound in the aqueous mixture, e.g., using a greater excess of peroxide compound relative to the manganese complex, such as a molar excess of > 2000.

[0148] At low pHs, the peroxide compound and / or ligand of formula (I) or (II) can be protonated. Consequently, the activation of the peroxide compound by the manganese complex can be inhibited and / or the ligand of formula (I) or (II) can dissociate from the manganese complex. Furthermore, biofilms are often less prone to degradation at low pHs. Inhibition of biofilm degradation can occur at a pH <4 or at a pH <3. Consequently, the pH of the aqueous mixture is typically from about 4 to about 12, more typically from about pH 6 to about 11. The pH of the aqueous medium is easily altered by the addition of acid or alkali (e.g., HCl or NaOH) or by using a buffered solution, i.e., in some embodiments, the aqueous mixture comprises a buffer. Often, a buffer is added to the aqueous mixture when the manganese complex is the product of a reducing agent and a manganese compound.Typically, the buffer is selected from the group consisting of carbonate, phosphate, or borate, preferably carbonate buffers. Often, the buffer is a carbonate, and the pH of the aqueous mixture is about 8 to about 10.5, often about 8 to about 10, typically about 8.5 to 9.5 and higher. Petition 870220076624, dated 08 / 25 / 2022, page 43 / 115 40 / 99 typically about 9. When added as a solution or suspension, the buffer is typically prepared by dissolving the solid buffer in a solvent (typically water). Alternatively, the buffer can be added to the aqueous mixture as a solid. The buffer can be added before, during, or with the manganese complex or compound, peroxide compound, and / or optional ligand of formula (I) or (II).

[0149] Alternatively, according to particular embodiments, a solid or liquid buffer is not added to the aqueous mixture. For the avoidance of doubt, such embodiments do not exclude the natural formation of a buffer in the aqueous mixture, for example, by the dissolution of carbon dioxide from the air in the aqueous mixture, thus forming a carbonate or bicarbonate buffer. According to other embodiments, no buffer is added to the aqueous mixture.

[0150] If the manganese complex is not the product of a reducing agent and a manganese compound, then the pH of the aqueous mixture is typically from about 9.5 to about 11.5, more typically from about 10 to about 11, and even more typically from about 10 to about 10.5. At these pH values, a sequestrant is typically included in the aqueous mixture. Sequestrants are described below.

[0151] The temperature of the aqueous mixture is typically from about 15 °C to about 90 °C, more typically from about 20 °C to about 70 °C. Preferably, the temperature of the aqueous mixture is from about 25 °C to about 50 °C.

[0152] The methods of the invention can be carried out for any length of time. Those skilled in the art are aware that a longer reaction time will lead to a greater degree of biofilm degradation. Even so, the reaction method effectively degrades the biofilm in reaction times of less than 10 minutes. Typically, the methods of the invention are carried out for about 0.1 to about 60 minutes, more typically about 0.1 to about 30 minutes, and preferably about 0.1 to about 10 minutes.

[0153] In contact with the biofilm, the degradation activity of the aqueous mixture of the first and second aspects of the invention may increase over time. Petition 870220076624, dated 08 / 25 / 2022, p. 44 / 115 41 / 99 Without being limited by theory, biofilm components can donate electrons to manganese complexes in the aqueous mixture, thus reducing the manganese complexes in a manner similar to the reducing agents mentioned above. Components that may be suitable for reducing manganese complexes in the aqueous mixture include proteins comprising amino acid residues that can act as reducing agents for manganese complexes in the aqueous mixture. Amino acid residues that may be suitable for such reduction include tyrosine and cysteine ​​residues. Low oxidation state metal ions, such as Fe(II), may be present in the biofilm and may also be suitable components for reducing manganese complexes in the aqueous mixture.

[0154] Reduced manganese complexes, i.e., those with less positive oxidation states, can be more active than the original manganese complexes. For example, a two-electron reducing component of the biofilm can reduce both Mn(IV) ions of a binuclear Mn(IV)Mn(IV) complex to produce a binuclear Mn(III)Mn(III) complex. Alternatively, poorly defined manganese complexes can be formed, for example, a mixture of binuclear Mn(II)Mn(II), Mn(III)Mn(II), Mn(III)Mn(III), Mn(III)Mn(IV), or Mn(IV)Mn(IV) complexes can form. It may also be possible for binuclear manganese complexes to split apart leading to the formation of mononuclear Mn complexes, i.e., Mn(II), Mn(III) and / or Mn(IV) complexes, see for example BC Gilberto et al. (above).

[0155] As mentioned in this document, the aqueous mixture of the first and second aspects comprises an optionally buffered solvent, which is typically water. However, other solvents may be used. The identity of the solvent is not an essential feature of the invention, provided that the solvent is miscible with water. The aqueous mixture will generally have at least 1% by weight of water, meaning that the water-containing liquid that Petition 870220076624, dated 08 / 25 / 2022, page 45 / 115 42 / 99 constitutes the aqueous liquid mixture comprising at least 1% by weight of water, more typically at least 10% by weight, even more typically 25% by weight, and most typically at least 50% by weight of water. The liquid equilibrium (if any) of the aqueous mixture that is not water may be any convenient liquid, for example, a liquid alcohol, e.g., a C1-C4 alcohol such as methanol or ethanol. Although the solvent is often entirely water, it will be understood that this does not exclude the presence of small amounts of other liquids (e.g., in a total amount less than about 10% by weight, more typically less than about 5% by weight), for example, as contaminants in other materials with which the continuous liquid phases are brought into contact.

[0156] In some embodiments, the aqueous mixture of the first and second aspects of the invention comprises a scavenger selected from the group consisting of an aminophosphate, an aminocarboxylate, and a carboxylate. When present, the scavenger is typically in concentrations of about 0.001 to about 10 g / l. Without being limited by theory, a scavenger may have two functions. Firstly, the scavenger may enhance the activity of the manganese complex (e.g., as disclosed in WO 2007 / 042192 A (Unilever PLC)), and / or it may bind to manganese impurities that may be present in the aqueous mixture or the biofilm. Manganese ions, such as Cu, Mn, or Fe, are well known to react with peroxide compounds and, in particular, with hydrogen peroxide to decompose the hydrogen peroxide into water and dioxygen or to form radicals such as superoxide and / or hydroxyl radicals.This can result in less peroxide compound available in the aqueous mixture to be activated by the manganese complex and thus may inhibit biofilm degradation. Therefore, the sequestrant may prevent the inhibition of biofilm degradation by manganese impurities present in the aqueous mixture and / or in the biofilm.

[0157] Preferred aminophosphonate sequestrants include nitrile trimethylene phosphonate, ethylenediamine-N,N,N',N'-tetra(methylene phosphonate) (Dequest 204TM) and Petition 870220076624, dated 08 / 25 / 2022, page 46 / 115 43 / 99 diethylenetriamine-N,N,N',N”,N”- penta(methylene phosphonate) (Dequest 206™). A person skilled in the art will be aware that there are different salts of each Dequest™, for example, as phosphonic acid or as sodium salts or any mixture thereof.

[0158] Preferred aminocarboxylate scavengers include ethylenediaminetetraacetic acid (EDTA), N-hydroxyethylenediaminetetraacetic acid (HEDTA), nitrilotriacetic acid (NTA), N-hydroxyethylaminodiacetic acid, N-hydroxyethylaminodiacetic acid, glutamic diacetic acid, sodium iminodisuccinate, diethylenetriaminepentacetic acid (DTPA), ethylenediamine-N,N'disuccinic acid (EDDS), methylglycinodiacetic acid (MGDA), and alanino-N,Ndiacetic acid.

[0159] The sequestrant may be in the form of a salt. For example, the sequestrant may comprise one or more cations selected from the group consisting of alkali metal ions, alkaline earth metal ions, ammonium ions or substituted ammonium ions. Preferably, the sequestrant is in the form of a free acid, or comprises sodium or magnesium cations, i.e., it is in its sodium or magnesium salt form.

[0160] Preferred carboxylate scavengers are polycarboxylates containing two carboxyl groups, including the water-soluble salts of succinic acid, malonic acid, (ethylenedioxy) diacetic acid, gluconic acid, maleic acid, diglycolic acid, tartaric acid, tartronic acid and fumaric acid, as well as ether carboxylates. Polycarboxylates containing three carboxyl groups include, in particular, water-soluble citrates, aconitrates and citraconates, as well as succinate derivatives such as carboxymethyloxysuccinates. Polycarboxylates containing four carboxyl groups include oxydisuccinates disclosed in GB 1261829 A (Unilever Ltd), 1,1,2,2-ethane tetracarboxylates, 1,1,3,3-propane tetracarboxylates and 1,1,2,3-propane tetracarboxylates.Polycarboxylates containing sulfo substituents include the sulfosuccinate derivatives disclosed in GB 1398421 A (Unilever Ltd) and GB 1398422 A (Unilever Ltd) and in US 3936448 A (Lever Brothers Ltd), and the described sulfonated pyrolyzed citrates. Petition 870220076624, dated 08 / 25 / 2022, page 47 / 115 44 / 99 in GB 1439000 A (Henkel & CIE GmbH).

[0161] Other suitable carboxylate scavengers are homo- or copolymeric polycarboxylic acids or their salts wherein the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by no more than two carbon atoms. Polymers of the latter type are described in GB 1596756 A (Proctor & Gamble Ltd). Examples of such salts are polyacrylates of molecular weight from 2,000 to 5,000 and their copolymers with maleic anhydride, such copolymers having a molecular weight from 20,000 to 70,000, especially around 40,000.

[0162] Also, copolymeric polycarboxylate polymers that, at least formally, are formed from an unsaturated polycarboxylic acid, such as maleic acid, citraconic acid, itaconic acid and mesaconic acid as the first monomer, and an unsaturated monocarboxylic acid, such as acrylic acid or an alpha-C1-C4 alkylacrylic acid as the second monomer. These polymers are available from BASF under the trade names Sokalan® CP5 (neutralized form), Sokalan® CP7 and Sokalan® CP45 (acid form).

[0163] Typically, the sequestrant is ethylenediamine-N,N,N',N'tetra(methylenephosphonate) (Dequest 204), diethylenetriamine-N,N,N',N",N"penta(methylenephosphonate) (Dequest 206), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), methylglycinediacetic acid (MGDA), acid citric, alkaline salts citrate and gluconate.

[0164] Often, the sequestrant is an ethylenediamine-N,N,N',N'tetra(methylene phosphonate) (Dequest 204). This is especially the case where the manganese complex of the first aspect of the invention is not the product of a manganese compound and a reducing agent.

[0165] The sequestrant is optionally present in the aqueous mixture comprising the transition metal complex, peroxide compound and ligand of formula (I) or (II). Alternatively, or additionally, the biofilm may be contacted with one or more of the aforementioned sequestrants prior to contact with the aqueous mixture. Without being limited by theory, if the biofilm Petition 870220076624, dated 08 / 25 / 2022, page 48 / 115 45 / 99 contains manganese impurities, and the aqueous mixture contains hydrogen peroxide, so contact of the biofilm with the scavenger before contact with the aqueous mixture can reduce the number of manganese impurities that could otherwise decompose the hydrogen peroxide. The removal of ions such as Ca2+ or Mg2+ can make the biofilm more susceptible to degradation.

[0166] When added as a solution or suspension, the sequestrant is typically prepared by dissolving the solid sequestrant in a solvent (typically water). Alternatively, the sequestrant may be added to the aqueous mixture as a solid. The sequestrant may be added before, during, or at the same time as the manganese complex, peroxide compound, optional ligand, and / or buffer.

[0167] The aqueous mixture of the first and second aspects may comprise additional agents that aid in biofilm degradation. The additional agents may be antimicrobial agents that aid in the degradation of microbial cells, thereby increasing biofilm degradation. The additional agents may inhibit biofilm growth.

[0168] The removal or partial removal of biofilm on a surface leads to fewer bacteria present on the surface, which consequently leads to slower growth of new biofilms.

[0169] In some embodiments, the biofilm comprises any one or a combination of polysaccharides. Without being limited by theory, the depolymerization of polysaccharides within the biofilm can lead to a weaker EPS matrix, which can then be cleaned or degraded more easily. Typically, the polysaccharide is any one or a combination of alginate, cholanic acid, dextran, kefiran, curdlan, welan, gellan, and xanthan gum. More typically, the biofilm comprises any one or a combination of alginate, dextran, kefiran, curdlan, welan, gellan, and xanthan gum. The polysaccharide is frequently an alginate. In certain embodiments, the alginate is produced by bacteria (preferably Azotobacter and / or Pseudomonas) or algae (from Petition 870220076624, dated 08 / 25 / 2022, page 49 / 115 46 / 99 preference for green algae).

[0170] In some embodiments, the biofilm is on the surface of a membrane; a pipe; other plumbing equipment; cleaning equipment (including laundry, dishwashers and bathroom equipment such as sinks, bathtubs, showers, dishwashers, washing machines, dryers, bidets); a toilet; a kitchen; a pantry; a changing room, for example in a gym or leisure center; walls and floors (and / or surfaces within spaces used for cleaning (e.g., shower walls and floors)); a cooling and / or heating system; a water vessel (including ship and boat hulls); and a marine apparatus.

[0171] The biofilm can, before contact with the aqueous mixture, be brought into contact with enzymes such as amylase and / or protease enzymes in order to degrade the sugars and proteins within the biofilm.

[0172] The methods of the invention can be carried out in batch and continuous processes and can be carried out, for example, in vessels, pipes and / or tubes. The aqueous mixture can be agitated or stirred during the method of the invention. This can increase the rate of biofilm degradation and / or allow larger quantities of biofilm to be degraded.

[0173] The methods of the invention can be carried out in conjunction with other processes that improve biofilm degradation. For example, the microwave-assisted depolymerization process described in WO 2014 / 102332 A1 (Dupont Nutrition Biosciences APS) can be carried out before, after, or simultaneously with the methods of the invention.

[0174] Any and all patent and non-patent references referred to herein are incorporated herein by reference in their entirety, as if the entire content of each reference were set forth herein in its entirety.

[0175] The invention may be further understood with reference to the following non-limiting clauses:

[0176] 1. A method for degrading a biofilm comprising contacting the biofilm with an aqueous mixture comprising (i) a compound of Petition 870220076624, dated 08 / 25 / 2022, p. 50 / 115 47 / 99 peroxide and (ii) a mononuclear complex of manganese Mn(II), Mn(III) or Mn(IV), or binuclear Mn(II)Mn(II), Mn(III)Mn(II), Mn(III)Mn(III), Mn(III)Mn(IV) or Mn(IV)Mn(IV), where the aqueous mixture comprises a ligand of formula (I) or (II): R2RiC R4R3C RN

[0177] where: R R4R3C R CR3R4 C---CRi R3 r2R4 CR-, R2 r2r^c CR3R4 Q' NR r3r4 R1 r2(II), --N-- [CR^CR^R.) Q = 1234

[0178] p is 3;

[0179] each R is independently selected from the group consisting of hydrogen, C1-C24 alkyl, CH2C6-Cwaryl, CH2CH2OH, CH2COOH, and pyridin-2-ylmethyl;

[0180] Q' is an ethylene or propylene bridge; and

[0181] R1, R2, R3 and R4 are independently selected from: H, C1-C4 alkyl and C1-C4 hydroxyalkyl.

[0182] 2. The method of clause 1, in which the manganese complex comprises the ligand.

[0183] 3. The method of clause 1 or clause 2, wherein the aqueous mixture comprises uncomplexed ligand of formula (I) or (II).

[0184] 4. The method of any of the preceding clauses, where each R Petition 870220076624, dated 08 / 25 / 2022, page 51 / 115 48 / 99 is independently selected from the group consisting of C1-C24-alkyl, CH2C6-C1-ioaryl, CH2CH2OH and CH2COOH.

[0185] 5. The method of any of clauses 1 to 3, wherein each R is independently selected from the group consisting of C1-C12 alkyl, CH2C6-C10 aryl, CH2CH2OH and CH2COOH.

[0186] 6. The method of any of clauses 1 to 3, wherein each R is independently selected from the group consisting of C1-C6 alkyl and benzyl.

[0187] 7. The method of any of the previous clauses, where each R is the same.

[0188] 8. The method of any of clauses 1 to 3, where each R is methyl.

[0189] 9. The method of any of the preceding clauses, where R1, R2, R3 and R4 are independently hydrogen or methyl.

[0190] 10. The method of any of clauses 1 to 8, where R1, R2, R3 and R4 are hydrogen.

[0191] 11. The method of any of the preceding clauses, where Q' is an ethylene bridge.

[0192] 12. The method of any of clauses 1 to 3, where the binder is Me3-TACN or Me4-DTNE.

[0193] 13. The method of any of the preceding clauses, wherein the molar ratio between the ligand of formula (I) and manganese is from about 100:1 to about 0.5:1.

[0194] 14. The method of any of clauses 1 to 12, wherein the molar ratio between the ligand of formula (I) and manganese is from about 10:1 to about 0.5:1.

[0195] 15. The method of any of clauses 1 to 12, wherein the molar ratio between the ligand of formula (I) and manganese is from about 5:1 to about 0.8:1.

[0196] 16. The method of any of clauses 1 to 12, where the reason Petition 870220076624, dated 08 / 25 / 2022, p. 52 / 115 The molar ratio between the ligand of formula (I) and manganese is about 2:1 to about 1.001:1.

[0197] 17. The method of any of the preceding clauses, wherein the molar ratio between the ligand of formula (II) and manganese is from about 50:1 to about 0.05:1.

[0198] 18. The method of any of clauses 1 to 16, wherein the molar ratio between the ligand of formula (II) and manganese is from about 5:1 to about 0.1:1.

[0199] 19. The method of any of clauses 1 to 16, wherein the molar ratio between the ligand of formula (II) and manganese is from about 3:1 to about 0.2:1.

[0200] 20. The method of any of clauses 1 to 16, wherein the molar ratio between the ligand of formula (II) and manganese is from about 1:1 to about 0.5001:1.

[0201] 21. The method of any of the preceding clauses, wherein the manganese complex is part of a salt comprising one or more non-coordination counterions selected from the group consisting of SO42-, R5COO-, Cl-, NO3-, R6SO3- and PFe-, where:

[0202] R5 is selected from the group consisting of hydrogen, C1-C12 alkyl and phenyl optionally substituted by C1-C12 alkyl; and

[0203] Reé selected from the group consisting of phenyl optionally substituted with C1-Cealkyl, C1-Cealkyl, and CF3.

[0204] 22. The method of clause 21, wherein R5 is selected from the group consisting of hydrogen, C1-Cealkyl and phenyl optionally substituted by C1-Cealkyl.

[0205] 23. The method of clause 21, where R5 is methyl or phenyl.

[0206] 24. The method of clause 21, where R5 is methyl.

[0207] 25. The method of any of clauses 21 to 24, wherein Reé phenyl optionally substituted with one or more methyl groups.

[0208] 2e. The method of any of clauses 21 to 24, where ReSO3- is Petition 870220076624, dated 08 / 25 / 2022, p. 53 / 115 50 / 99 tosylate.

[0209] 27. The method of clause 21, in which non-coordination counterions are selected from the group consisting of acetate, chloride, sulfate, nitrate and hexafluorophosphate.

[0210] 28. The method of clause 21, in which non-coordination counterions are selected from the group consisting of acetate, chloride, sulfate and nitrate.

[0211] 29. The method of any of the preceding clauses, in which the manganese complex is binuclear.

[0212] 30. The method of clause 29, wherein the binuclear manganese complex comprises two or three independently selected bridging ligands from the group consisting of oxide, hydroxide, water, phenylboronate and R5COO-;

[0213] wherein R5 is selected from the group consisting of hydrogen, C1-C12 alkyl and phenyl optionally substituted by C1-C6 alkyl.

[0214] 31. The method of clause 30, wherein R5 is selected from the group consisting of hydrogen, C1-C12 alkyl and phenyl optionally substituted with one or more methyl groups.

[0215] 32. The method of clause 30, wherein R5 is selected from the group consisting of hydrogen, C1-C6 alkyl and phenyl.

[0216] 33. The method of clause 30, where R5 is methyl or phenyl, for example, methyl.

[0217] 34. The method of clause 29, in which the binuclear manganese complex comprises two or three bridging ligands selected independently from among oxide, hydroxide, water, acetate and benzoate.

[0218] 35. The method of any of clauses 29 to 34, wherein the binuclear manganese complex comprises three bridging ligands.

[0219] 36. The method of any of clauses 29 to 35, wherein the binuclear manganese complex is a complex of Mn(III)Mn(III), Mn(III)Mn(IV) or Mn(IV)Mn(IV).

[0220] 37. The method of any of clauses 1 to 28, wherein the manganese complex is any of the group consisting of [MnIIIMnIII(qO)(qPetition 870220076624, of 25 / 08 / 2022, p. 54 / 115 51 / 99 R5COO)2(Me3-TACN)2]2+, [MnIIIMnIV(pO)(p-R5COO)2(Me3-TACN)2]3+, [ΜηινΜηιν(μ-Ο)3(Μθ3-ΤΑΟΝ)2]2+, [MnIIIMnIV(ii-O)2(ii-R5COO)(Me4-DTNE)]2+and [MnIVMnIV(pO)2(p-R5COOD)(Me4-NE) was selected. between hydrogen, Ci-Ci2alkyl and optionally Ci-Cealkyla-substituted phenyl.

[0221] 38. The method of any one of clauses 1 to 28, wherein the manganese complex is part of a salt, wherein the salt is any one of the group comprising [MnIIIMnIII(pO)(p-R5COO)2(Me3-TACN)2][CH3COO]2, [MnIIIMnIV(pO)(p-R5COO)2(Me3-TACN)2][CH3COO]3, [MnIVMnIV(pO)3(Me3TACN)2][CH3COO]2, [MnIIIMnIII(pO)(p-R5COO)2(Me3-TACN)2][SO4] [MnIIIMnIV(pO)(p-R5COO)2(Me3-TACN)2]2[SO4]3, [MnIVMnIV(pO)3(Me3TACN)2][SO4], [MnIIIMnIII(pO)(p-R5COO)2(Me3-TACN)2][NO3]2, [MnIHMnIV(pO)(p-R5COO)2(Me3-TACN)2][NO3]3, [MnIVMnIV(pO)3(Me3-TACN)2][NO3]2, [MnIVMnIV(pO)3(Me3-TACN)2][PF6]2, [MnIIIMnIV(pO)2(p-R5COO)(Me4DTNE)][Cl]2 and [MnIVMnIV(pO)2(p-R5COO)(Me4-DTNE)][Cl]3, wherein R5 is selected from hydrogen, Ci-Ci2alkyl and phenyl optionally substituted by Ci-Cealkyl.

[0222] 39. The method of either clause ia 28, wherein the manganese complex is [MnIVMnIV(pO)3(Me3-TACN)2]2+ or [MnIIIMnIV(pO)2(pR5COO)(Me4-DTNE)]2+, wherein R5 is selected from hydrogen, C1-C12 alkyl and phenyl optionally substituted by C1-C12 alkyl.

[0223] 40. The method of any of clauses 37 to 39, where R5 is methyl.

[0224] 4i. The method of any of clauses ia 28, wherein the manganese complex is part of a salt, wherein the salt is [MnIVMnIV(pO)3(Me3TACN)2][CH3COO]2, [MnIVMnIV(pO)3(Me3-TACN)2][SO4], [MnIVMnIV(pO)3(Me3TACN)2][NO3]2, [MnIVMnIV(pO)3(Me3-TACN)2][PFe]2, or [MnIIIMnIV(pO)2(pCH3COO)(Me4-DTNE)][Cl]2.

[0225] 42. The method of any of clauses ia 36, ​​in which, before contact, a manganese compound comes into contact with a reducing agent to provide the manganese complex. Petition 870220076624, dated 08 / 25 / 2022, p. 55 / 115 52 / 99

[0226] 43. The method of clause 42, wherein the reducing agent is selected from the group consisting of ascorbic acid, ascorbyl palmitate, ascorbyl stearate, catechol, 4-tert-butyl-catechol, 4-allylcatechol, caffeic acid, maltol, ethylmaltol, hydroquinone, tert-butyl-hydroquinone, 2,5-di-tert-butyl-hydroquinone, pyrogallol, and n-propyl gallate, an alkali metal sulfite, an alkali metal bisulfite and an alkali metal thiosulfate.

[0227] 44. The method of clause 42, in which the reducing agent is selected from the group consisting of ascorbic acid, catechol, hydroquinone, pyrogallol, and sodium sulfite.

[0228] 45. The method of clause 42, wherein the reducing agent is ascorbic acid.

[0229] 46. The method of any of clauses 42 to 45, wherein the molar ratio between the manganese compound and the reducing agent is from about 0.1:1 to about 10:1.

[0230] 47. The method of any of clauses 42 to 45, wherein the molar ratio between the manganese compound and the reducing agent is from about 0.2:1 to about 3:1.

[0231] 48. The method of any of clauses 42 to 47, wherein the manganese compound comprises a non-coordination contraion selected from the group consisting of SO42-, R5COO-, Cl-, NO3-, R6SO3- and PF6-, wherein:

[0232] R5 is selected from the group consisting of hydrogen, C1-C12 alkyl and phenyl optionally substituted by C1-C6 alkyl; and

[0233] R6 is selected from the group consisting of phenyl optionally substituted with C1-C6 alkyl, C1-C6 alkyl, and CF3.

[0234] 49. The method of clause 48, wherein R5 is selected from the group consisting of hydrogen, C1-C6 alkyl and phenyl optionally substituted by C1-C6 alkyl.

[0235] 50. The method of clause 48, where R5 is methyl or phenyl.

[0236] 51. The method of clause 48, where R5 is methyl.

[0237] 52. The method of any of the clauses 48 to 51, where R6 is phenyl Petition 870220076624, dated 08 / 25 / 2022, p. 56 / 115 53 / 99 optionally replaced with one or more methyl groups.

[0238] 53. The method of any of clauses 48 to 52, where R6SO3- is tosylate.

[0239] 54. The method of clause 48, in which the non-coordination contraion is selected from the group consisting of acetate, chloride, sulfate, nitrate and hexafluorophosphate.

[0240] 55. The method of clause 48, in which the non-coordination contraion is selected from the group consisting of acetate, chloride, sulfate and nitrate.

[0241] 56. The method of any of clauses 42 to 55, wherein the manganese compound comprises a ligand of formula (I) or (II): (Q)p CR-| R2 (I) CR3R4 (II),

[0242] where: ---N--- [CR1R2CR3R4)

[0243] p is 3;

[0244] each R is independently selected from the group consisting of hydrogen, C1-C24alkyl, CH2C6-Cwaryl, CH2CH2OH, CH2COOH, and pyridin-2ylmethyl;

[0245] Q' is an ethylene or propylene bridge; and

[0246] R1, R2, R3 and R4 are independently selected from: H, C1 Petition 870220076624, dated 08 / 25 / 2022, page 57 / 115 54 / 99 C4alkyl and C1-C4hydroxyalkyl.

[0247] 57. The method of clause 56, wherein each R of the manganese compound is independently selected from the group consisting of C1-C24 alkyl, CH2C6-C10 aryl, CH2CH2OH and CH2COOH.

[0248] 58. The method of clause 56, wherein each R of the manganese compound is independently selected from the group consisting of C1-C12 alkyl, CH2C6-C10 aryl, CH2CH2OH and CH2COOH.

[0249] 59. The method of clause 56, wherein each R of the manganese compound is independently selected from the group consisting of C1-C6 alkyl and benzyl.

[0250] 60. The method of any of clauses 56 to 59, where each R of the manganese compound is the same.

[0251] 61. The method of any of clauses 56 to 59, wherein each R of the manganese compound is methyl.

[0252] 62. The method of any of clauses 56 to 61, wherein R1, R2, R3 and R4 of the manganese compound are independently hydrogen or methyl.

[0253] 63. The method of any of clauses 56 to 61, where R1, R2, R3 and R4 of the manganese compound are hydrogen.

[0254] 64. The method of any of clauses 56 to 61, where Q' of the manganese compound is an ethylene bridge.

[0255] 65. The method of clause 56, in which the ligand of the manganese compound is Me3-TACN or Me4-DTNE.

[0256] 66. The method of any of clauses 42 to 65, wherein the manganese compound is a binuclear compound of Mn(III)Mn(III), Mn(III)Mn(IV) or Mn(IV)Mn(IV).

[0257] 67. The method of any of clauses 42 to 65, wherein the manganese complex is any one or a combination selected from the group consisting of mononuclear complexes of Mn(II), Mn(III) and Mn(IV), and binuclear complexes of Mn(II)Mn(II), Mn(III)Mn(II), Mn(III)Mn(III) and Mn(III)Mn(IV), and the manganese compound is selected from the group consisting of binuclear compounds of Petition 870220076624, dated 08 / 25 / 2022, p. 58 / 115 55 / 99 Mn(III)Mn(III), Mn(III)Mn(IV) and Mn(IV)Mn(IV).

[0258] 68. The method of clause 66 or clause 67, wherein the binuclear manganese compound comprises 2 or 3 bridging ligands independently selected from the group consisting of oxide, hydroxide, water, phenylboronate and R5COO-, wherein R5 is selected from the group consisting of hydrogen, C1-C12 alkyl and phenyl optionally substituted with C1-C6 alkyl.

[0259] 69. The method of clause 68, wherein R5 of the manganese compound is selected from the group consisting of hydrogen, C1-C12 alkyl and phenyl optionally substituted with one or more methyl groups.

[0260] 70. The method of clause 68, wherein R5 of the manganese compound is selected from the group consisting of hydrogen, C1-C6 alkyl and phenyl.

[0261] 71. The method of clause 68, wherein R5 of the manganese compound is selected between methyl and phenyl, for example, methyl.

[0262] 72. The method of clause 66 or clause 67, wherein the binuclear manganese compound comprises 2 or 3 bridging ligands selected independently from among oxide, hydroxide, water, acetate and benzoate.

[0263] 73. The method of clause 66 or clause 67, wherein the binuclear manganese compound comprises 3 bridging ligands selected independently from oxide, hydroxide, water, acetate and benzoate.

[0264] 74. The method of any of clauses 42 to 55, wherein the manganese compound comprises any of the group consisting of [MnIIIMnIII(pO)(p-R5COO)2(Me3-TACN)2]2+, [MnIIIMnIV(pO)(p-R5COO)2(Me3TACN)2]3+, [MnIVMnIV(pO)3(Me3-TACN)2]2+, [MnmMnIV(pO)2(p-R5COO)(Me4DTNE)]2+ and [MnIVMnIV(pO)2(p-R5COO)(Me4-DTNE)]3+, wherein R5 is selected from hydrogen, C1-C12 alkyl and phenyl optionally substituted by C1-C12 alkyl.

[0265] 75. The method of any one of clauses 42 to 47, wherein the manganese compound is any one of the group comprising [MnIIIMnIII(pO)(p-R5COO)2(Me3-TACN)2][CH3COO]2, [MnIHMnIV(pO)(p-R5COO)2(Me3Petition 870220076624, dated 25 / 08 / 2022, p. 59 / 115 56 / 99 TACN)2][CH3COO]3, [ΜηινΜπιν(μ-0)3(Μθ3-ΤΑ0Ν)2][0Η3000]2, [ΜηιιιΜηιιι(μC)2[TA2][Me2]R5CO [Μη^Μη^μ-ΟΧμ^ΟΟ^^TACN)2]2[SO4]3, [MnIVMnIV(μ-O)3(Me3-TACN)2][SO4], [Μη^Μη'^^)^R^OO^^-TACN^NO^, [MnIIIMnIV(μ-O)(μ-R5COO)2(Me3-TACN)2][NO3]3, [MnIVMnIV(μ-O)3(Me3-TACN)2][NO3]2, [MnIVMnIV(μ-O)3(Me3-TACN)2][PF6]2, [MnIIIMnIV(µ-O)2(µ-R5COO)(Me4-DTNE)][Cl]2 and [MnIVMnIV(µ-O)2(µ-R5COO)(Me4DTNE)][Cl]3, so that R5é βθίΙοίοηγθόο ήίόΓΘ9έηίο, Ci-Cy2alkylate and fethyl οροιοηαίπθηίθ substituted by Ci-C6alkylate.

[0266] 76. The method of any of clauses 42 to 55, wherein the manganese compound is [MnIVMnIV(μ-O)3(Me3-TACN)2]2+ or ^^^^^^)2^R5COO)(Me4-DTNE)]2+, wherein R5 is selected from between hydrogen, C1-C12 alkyl and phenyl optionally substituted for C1-C6 alkyl.

[0267] 77. The method of any of clauses 74 to 76, wherein R5 of the manganese compound is methyl.

[0268] 78. The method of any of clauses 42 to 47, where the manganese compound is [MnIVMnIV(μ-O)3(Me3-TACN)2][CH3COO]2, [MnIVMnIV(μ-O)3(Me3-TACN)2][SO4], [MnIVMnIV(μ-O)3(Me3-TACN)2][NO3]2, [MnIVMnIV(μ-O)3(Me3-TACN)2][PF6]2, or [MnIIIMnIV(μ-O)2(μ-CH3COO)(Me4DTNE)][Cl]2.

[0269] 79. The method of any of the arteries clauses, in which the concentration of the manganese complex in the aqueous mixture is about 0.000ia about i00 μM.

[0270] 80. The method of any of the clauses ia 78, in which the concentration of the manganese complex in the aqueous mixture is from about 0.00i to about 50 μM.

[0271] 8i. The method of any of the clauses ia 78, where the concentration of the manganese complex in the aqueous mixture is from about 0.0i to about 30 μM.

[0272] 82. The method of any of the clauses ia 78, where the concentration of the manganese complex in the aqueous mixture is about 0.05 Petition 870220076624, dated 08 / 25 / 2022, pp. 60 / 115 57 / 99 at approximately 20 μm.

[0273] 83. The method of any of the preceding clauses, wherein the peroxide compound is any one or a combination of the group consisting of hydrogen peroxide, a peroxyacid, an alkyl hydroperoxide, a phenyl alkyl hydroperoxide, and a ketone peroxide.

[0274] 84. The method of any of clauses 1 to 82, wherein the peroxide compound is any one or a combination of the group consisting of hydrogen peroxide, a peroxyacid, C1-12 alkyl hydroperoxide and cumene hydroperoxide.

[0275] 85. The method of any of clauses 1 to 82, wherein the peroxide compound is a mixture of a peroxyacid and hydrogen peroxide.

[0276] 86. The method of clause 85, in which the molar ratio between peroxyacid and hydrogen peroxide is from about 10:1 to about 1:100.

[0277] 87. The method of clause 85, in which the molar ratio between peroxyacid and hydrogen peroxide is from about 5:1 to about 1:10.

[0278] 88. The method of any of clauses 83 to 87, wherein the peroxyacid is peracetic acid.

[0279] 89. The method of any of clauses 1 to 82, wherein the peroxide compound is a mixture of C1-12 alkyl hydroperoxide and hydrogen peroxide.

[0280] 90. The method of clause 89, wherein the molar ratio between C112 alkyl hydroperoxide and hydrogen peroxide is from about 10:1 to about 1:10.

[0281] 91. The method of any of clauses 89 to 90, wherein C12alkylhydroperoxide is tert-butylhydroperoxide.

[0282] 92. The method of any of the preceding clauses, wherein the concentration of the peroxide compound is from about 0.01 to about 500 mM.

[0283] 93. The method of any of clauses 1 to 91, wherein the concentration of the peroxide compound is from about 0.1 to about 100 mM.

[0284] 94. The method of any of clauses 1 to 91, wherein the concentration of the peroxide compound is from about 0.3 to about 30 mM. Petition 870220076624, dated 08 / 25 / 2022, pp. 61 / 115 58 / 99

[0285] 95. The method of any of the preceding clauses, wherein the temperature of the aqueous mixture is from about 15 °C to about 90 °C.

[0286] 96. The method of any of clauses 1 to 94, wherein the temperature of the aqueous mixture is from about 20 °C to about 70 °C.

[0287] 97. The method of any of the preceding clauses, wherein the pH of the aqueous mixture is from about 4 to about 12.

[0288] 98. The method of any of clauses 1 to 96, where the pH of the aqueous mixture is from about 6 to about 11.

[0289] 99. The method of any of the preceding clauses, wherein the aqueous mixture additionally comprises one or more sequestrants selected from the group consisting of an aminophosphonate, an aminocarboxylate and a carboxylate.

[0290] 100. The method of clause 99, wherein the aminophosphonate sequestrant is any one or a combination of the group consisting of nitrile trimethylene phosphonate, ethylenediamino-N,N,N',N'-tetra(methylene phosphonate) (Dequest 204TM) and diethylenetriamine-N,N,N',N”,N”-penta(methylene phosphonate) (Dequest 206TM); the aminocarboxylate sequestrant is any one of the group consisting of ethylenediaminetetraacetic acid, N-hydroxyethylenediaminetetraacetic acid, nitriloacetic acid, N-hydroxyethylaminoacetic acid, N-hydroxyethylaminoacetic acid, glutamic diacetic acid, sodium iminodisuccinate, diethylenetriaminopentacetic acid, ethylenediamino-N,N'-disuccinic acid, methylglycinodiacetic acid and alanine-N,N-diacetic acid; And the carboxylate sequestrant is any one of the group consisting of citric acid, alkaline citrate salts, and gluconate.

[0291] 101. The method of clause 99, wherein the aminophosphonate sequestrant is ethylenediamine-N,N,N',N'-tetra(methylene phosphonate) (Dequest 204TM) or diethylenetriamine-N,N,N',N”,N”-penta(methylene phosphonate) (Dequest 206TM); the aminocarboxylate sequestrant is selected from ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, and methylglycinodiacetic acid; and the carboxylate sequestrant is selected from acid Petition 870220076624, dated 08 / 25 / 2022, page 62 / 115 59 / 99 citric acid, alkaline salts of citrate and gluconate.

[0292] 102. The method of any of the preceding clauses, wherein the aqueous mixture comprises a buffer.

[0293] 103. The method of clause 102, wherein the buffer is any one or a combination selected from the group consisting of phosphate, carbonate and borate.

[0294] 104. The method of clause 102, in which the buffer is a carbonate.

[0295] 105. The method of clause 102, where the pH of the aqueous mixture is from about 8 to about 10.5.

[0296] 106. The method of clause 102, where the pH of the aqueous mixture is from about 8 to about 10.

[0297] 107. The method of clause 102, where the pH of the aqueous mixture is about 8.5 to 9.5.

[0298] 108. The method of clause 102, where the pH of the aqueous mixture is about 9.

[0299] 109. The method of any of clauses 1 to 101, wherein a buffer is not added to the aqueous mixture.

[0300] 110. The method of clause 109, where the pH of the aqueous mixture is from about 9.5 to about 11.5.

[0301] 111. The method of clause 109, where the pH of the aqueous mixture is from about 10 to about 10.5.

[0302] 112. The method of any of the preceding clauses, wherein the biofilm comprises any one or a combination of constituents selected from the group consisting of alginate, bacterial cellulose, colonic acid, dextran, kefiran, curdlan, wedlan, gellan, and xanthan.

[0303] 113. The method of any of clauses 1 to 111, wherein the biofilm comprises alginate.

[0304] 114. The method of clause 112 or 113, in which the alginate is produced by bacteria or algae.

[0305] 115. The method of clause 114, in which the bacteria are Azotobacter and Pseudomonas. Petition 870220076624, dated 08 / 25 / 2022, p. 63 / 115 60 / 99

[0306] 116. The method of clause 114 or clause 115, wherein the algae are green algae.

[0307] 117. The method of any of the preceding clauses, wherein the method reduces the biofilm mass by at least 1% by weight.

[0308] 118. The method of any of the clauses 1 to 116, wherein the method reduces the biofilm mass by at least 10%.

[0309] 119. A method for degrading a biofilm comprising contacting the biofilm with an aqueous mixture comprising a peroxide compound and a binder of formula (I) or (II): R ^-N R4R3C R R2R-|C CR3R4 CR·] R2 R4R3C CR-, R2 R2R-jC CR3R4 RN Q' NR C---CR1 R3r2r4 R3r4 R1 R2(II),

[0310] where: R I ---N--- [CR1R2CR3R4)

[0311] p is 3;

[0312] each R is independently selected from the group consisting of hydrogen, C1-C24-alkyl, CH2C6-Cwaryl, CH2CH2OH, CH2COOH, and pyridin-2-ylmethyl;

[0313] Q' is an ethylene or propylene bridge; and

[0314] R1, R2, R3 and R4 are independently selected from: H, C1-C4 alkyl and C1-C4 hydroxyalkyl. Petition 870220076624, dated 08 / 25 / 2022, page 64 / 115 61 / 99

[0315] EXPERIMENTAL PART

[0316] Raw materials

[0317] · Me3-TACN was obtained as disclosed in document WO 94 / 08981 A1.

[0318] · [Mn2(DO)3(Me3-TACN)2](CH3COO)2 (as a 3.5 wt% aqueous solution in acetate buffer pH 5, made from 2.4 wt% Na-acetate, 1.8 wt% glacial acetic acid and adjusted to pH 5) was obtained as disclosed in document WO 2006 / 125517 A1.

[0319] · [Mn2(DO)2(D-CH3COO)(Me4DTNE)]Cl2 was prepared as disclosed in document WO 2011 / 106906 A1.

[0320] · Sodium alginate was purchased from BDH Prolabo (WVR).

[0321] · All other chemicals were obtained from standard chemical suppliers.

[0322] Preparation of the initial alginate solution

[0323] First, a 1.5% by weight alginate solution in water was prepared by slowly adding 7.5 g of solid sodium alginate to 492.5 g of demineralized water while vigorously stirring using a mechanical stirrer. The mixture was left to stir at room temperature (which here denotes 20 °C) until no more solid sodium alginate was visible.

[0324] Stock solutions

[0325] - Stock 1: A 5 M stock solution of hydrogen peroxide was prepared by placing 4.29 mL of commercial hydrogen peroxide (35% purity) in a 10 mL volumetric flask and completing with demineralized water until it reached the 10 mL mark.

[0326] - Stock 2: A 0.5 M stock solution of tert-butyl hydroperoxide (abbreviated as tBuOOH) was obtained by placing 0.685 mL of a 70% purity commercial solution of tBuOOH in a 10 mL volumetric flask and making up to the 10 mL mark with demineralized water.

[0327] - Stock 3: A stock solution of 39.6 g / L NaHCO3 was obtained by weighing 3.96 g of solid NaHCO3 powder and placing it in a 100 mL glass beaker. Approximately 50 mL of demineralized water was added and the mixture was stirred. Petition 870220076624, dated 08 / 25 / 2022, pp. 65 / 115 62 / 99 the solution until the powder was completely dissolved. Once dissolved, the pH was adjusted to pH 7.4 using diluted HCl before the contents of the beaker were transferred to a 100 mL volumetric flask. The beaker was washed with demineralized water and the washing medium was also transferred to the flask. Finally, the volumetric flask containing the carbonate solution was filled with demineralized water to the 100 mL mark and the contents were shaken.

[0328] - Stock 4: A stock solution of 25 g / L sodium tetraborate decahydrate was prepared by placing 2.5 g of the solid powder in a 100 mL volumetric flask. Demineralized water was then added and the flask was shaken until the solid was completely dissolved. Finally, demineralized water was added until the 100 mL demineralized mark was reached.

[0329] - Stock 5: 5 mM ascorbic acid: 17.62 mg of commercial L-ascorbic acid were weighed and placed in a 20 mL volumetric flask. Demineralized water was added to the flask until it reached the 20 mL mark. The flask was then closed and shaken until the solid was completely dissolved.

[0330] - Stock 6: 0.5 mM neutralized ascorbic acid: 2 mL of the 5 mM ascorbic acid stock solution were placed in a 20 mL flask followed by 15 mL of demineralized water. The solution was stirred and the pH was measured. 0.1 M NaOH was added to bring the pH of the solution to pH 6-7 (the exact volume of NaOH used was recorded). Finally, the volume of the solution was increased to 20 mL by adding demineralized water.

[0331] - Stock 7: A 2 mM stock solution of neutralized L-ascorbic acid was prepared as follows: 35.22 mg of ascorbic acid powder were weighed and transferred to a 100 mL glass beaker. Approximately 60 mL of demineralized water were added before the solution was stirred. Once the solid was completely dissolved, the diluted NaOH solution was used to bring the pH of the solution from pH 6 to 7 before the solution was transferred to a 100 mL volumetric flask. The beaker was washed with demineralized water, the wash water was transferred to the volumetric flask, and finally the volume of the solution was increased to 100 mL using water. Petition 870220076624, dated 08 / 25 / 2022, p. 66 / 115 63 / 99 demineralized.

[0332] - Stock 8: A 4 mM stock solution of manganese sulfate was made by dissolving 13.52 mg of manganese sulfate monohydrate in a 20 mL volumetric flask using demineralized water. The total volume of the solution was 20 mL.

[0333] - Stock 9: A 4 mM stock solution of manganese chloride was made by dissolving 15.83 mg of MnCl2.4H2O in a 20 mL volumetric flask using demineralized water. The total volume of the solution was 20 mL.

[0334] - Stock 10: A 4 mM Me3-TACN stock solution was obtained by placing 14.43 mg of a 95% pure commercial solution of the product in a 20 mL volumetric flask. Approximately 18 mL of demineralized water were added followed by 80 DL of HCl (1 M) before the solution was shaken. Finally, demineralized water was added until the 20 mL mark was reached. The flask was stoppered and shaken until the solution was homogeneous.

[0335] - Stock 11: 10 mL of a 2 mM aqueous solution of [Mn2(DO)2(DCH3COO)(Me4DTNE)]Cl2 were prepared by dissolving 24.24 mg of a 50.5% pure commercial batch of the catalyst in a 10 mL volumetric flask with demineralized water. Water was added until the 10 mL mark was reached, then the flask was stoppered and shaken until the solution was homogeneous.

[0336] - Stock 12: A 2 mM stock solution of [Mn2(DO)3(Me3TACN)2](CH3COO)2 was obtained by diluting 0.327 mL of a 3.5% by weight solution with 9.573 mL of demineralized water in a 10 mL volumetric flask.

[0337] - Stock 13: A 0.5 mM stock solution of [Mn2(DO)3(Me3TACN)2](CH3COO)2 was obtained by diluting 0.163 mL of a 3.5% by weight solution with 19.827 mL of demineralized water in a 20 mL volumetric flask.

[0338] Procedure 1 Intrinsic Viscosity Measurements

[0339] Equipment and principle

[0340] Intrinsic viscosity was measured using a tube viscometer. Petition 870220076624, dated 08 / 25 / 2022, page 67 / 115 64 / 99 This is a type C manual SCAN viscometer acquired from PSL-Rheotek. It features a built-in water jacket connected to a circulating water bath at 25°C to maintain a constant temperature during testing.

[0341] The analysis consisted of measuring the time required for a solution to flow through the tube (efflux time in seconds); the efflux time is related to the viscosity of the sample. To make the measurements more reliable, the viscometer was equipped with two markings (upper and lower) delimiting a defined volume within the tube. Before each measurement, the viscometer was filled with the test solution to a level slightly above the upper mark, and the solution was kept in the tube for a few minutes to allow for temperature homogenization.

[0342] Next, the tube was opened to allow the liquid to flow. When the meniscus crossed the upper mark of the tube, the timer was started and when the liquid level reached the lower mark the stopwatch was stopped. The value displayed on the stopwatch corresponded to the efflux time. For greater reliability, the measurement was repeated a second time and the average efflux time was calculated.

[0343] Note that 0.1 mM NaCl was used as the solvent; the efflux time for this solution was 53.26 seconds.

[0344] Calculation of Intrinsic Viscosity

[0345] The intrinsic viscosity of a solution was obtained using Huggins' equation: ^r = 1η] + k'. [η]2. C (equation 1)

[0346] With:

[0347] - [rç] Intrinsic Viscosity in dL / g

[0348] - C: biofilm concentration in solution expressed in g / dL

[0349] - ηερa specific viscosity defined as: ηερ=Vsolution- 1 ^solvent

[0350] Since very dilute solutions were used, it can be assumed that the densities of the solvent and the test solutions were similar and simplify Petition 870220076624, dated 08 / 25 / 2022, p. 68 / 115 65 / 99 the equation is as follows: ^ = Time of efflux / flow? ão_ 1 (equation 2)pTime of effluxSoivente

[0351] From equation 1, the Intrinsic Viscosity of a sample can be obtained by diluting said sample in different biofilm concentrations before measuring the efflux time of the diluted solutions. Then, ^7 can be calculated for each concentration and plotted against the biofilm level (in g / dL) actually present. The data points obtained formed a straight line (see example in Fig. 1) with the Y-intercept corresponding to the Intrinsic Viscosity.

[0352] Calculation of the average molecular weight of alginate chains

[0353] The average molecular weight of the alginate chains was calculated using the Mark-Houwink equation. In this case, the values ​​for the different constants of the equations were obtained from Rheological Evaluation of Inter-grade and Inter-batch Variability of Sodium Alginate published in AAPS Pharm.Sci.Tech., Vol. 11, No. 4, December 2010. --(-)94 (equation 3) = (A 9 (equation 4)

[0354] As explained above, [rç] corresponds to the Intrinsic Viscosity in dL / g. The calculated average Molecular Weights are expressed in kilo Daltons (kDa).

[0355] Treatment of alginate solution

[0356] 200 mL of 1.5% by weight alginate solution were placed in a 500 mL glass beaker followed by 0.43 mL of H2O2 stock solution and 4.1 mL of demineralized water. The solution was homogenized using a magnetic stirrer and 0.37 mL of NaOH (1M) was added to bring the pH to pH 10.5. Then, 19.98 g aliquots of the alginate mixture were transferred to mL HDPE containers (Duma Container Special purchased from VWR).

[0357] 4 mL of [Mn2(qO)3(Me3-TACN)2](CH3COO)2 solution (0.5 mM, Petition 870220076624, dated 08 / 25 / 2022, page 69 / 115 66 / 99 stock 13) was premixed with 4 mL of 0.5 mM ascorbic acid solution (stock 6) in a 20 mL vial. The resulting sample was shaken until homogeneous before being used to dose the catalyst into the HDPE vials. The catalyst concentration in the samples ranged from 0 to 10 gM.

[0358] Finally, demineralized water was added to each bottle to bring the solution volume to 21 mL (i.e., the alginate content of the samples was 1.4% by weight). The bottles were closed and shaken to homogenize the systems. Immediately afterwards, the bottles were stored in a water bath heated to 50 °C for 60 minutes.

[0359] At the end of the reaction time, the bottles were removed from the water bath and placed in ice to stop the reaction.

[0360] Intrinsic viscosity measurements:

[0361] All analyses were performed using 0.1M NaCl as solvent. The intrinsic viscosity of 6 solutions was measured:

[0362] - Sample 1: 1.4% alginate, untreated

[0363] - Sample 2: 1.4% alginate treated with 10 mM H2O2

[0364] - Sample 3: 1.4% alginate treated with 10 mM H2O2 and 0.5 gM [Mn2(gO)3(Me3TACN)2](CH3COO)2 premixed with ascorbic acid (see description above)

[0365] - Sample 4: 1.4% alginate treated with 10 mM H2O2 eg [Mn2(gO)3(Me3-TACN)2](CH3COO)2 1 M premixed with ascorbic acid (see description above)

[0366] - Sample 5: 1.4% alginate treated with 10 mM H2O2 eg [Mn2(gO)3(Me3-TACN)2](CH3COO)2 2 M premixed with ascorbic acid (see description above)

[0367] - Sample 6: 1.4% alginate treated with 10 mM H2O2 eg [Mn2(gO)3(Me3-TACN)2](CH3COO)2 10 M premixed with ascorbic acid (see description above)

[0368] Each solution analyzed was diluted to three different alginate levels, as indicated in Table 1. Petition 870220076624, dated 08 / 25 / 2022, pp. 70 / 115 67 / 99

[0369]

[0370] Table below, using demineralized water and NaCl (1 M). All diluted solutions contained 0.1 M NaCl. Table 1: Overview of alginate concentrations in 0.1 M NaCl used for intrinsic viscosity measurements. Sample No. Original Alginate Content Alginate Content Dilution 1 Alginate Content Dilution 2 Alginate Content Dilution 3 1 1.4% 0.075% 0.1% 0.125% 2 1.4% 0.075% 0.1% 0.125% 3 1.4% 0.075% 0.1% 0.125% 4 1.4% 0.1% 0.15% 0.2% 5 1.4% 0.1% 0.2% 0.3% 6 1.4% 0.3% 0.5% 0.7%

[0371] The efflux time for each of the solutions was measured twice before the average was calculated. This value was used to calculate η3ρ, as explained above, considering an efflux time for the solvent equal to 53.26 seconds.

[0372] After plotting with respect to C (C = alginate content in g / dL), a linear regression was performed with the Y-intercept of the trend line corresponding to the Intrinsic Viscosity in dL / g. This value was used to calculate the average molecular weight of the polymer chains in the tested sample.

[0373] Procedure 2 Dynamic Viscosity Measurements

[0374] Treatment of alginate solution

[0375] 19.5 mL of 1.5% by weight alginate solution were placed in a plastic bottle followed by the oxidant stock solution (hydrogen peroxide Petition 870220076624, dated 08 / 25 / 2022, pp. 71 / 115 68 / 99 hydrogen, tert-butyl hydroperoxide, peracetic acid or mixtures thereof), optionally the sequestering agent and / or buffer, NaOH to adjust the pH and (when necessary) [Mn2(pO)3(Me3-TACN)2](CH3COO)2, [Mn2(pO)2(pCH3COO)(Me4DTNE)]Cl2, [Mn2(pO)3(Me3-TACN)2](CH3COO)2 premixed with 1 molar equivalent of ascorbic acid solution note 1 or MnSO4.H2O mixed with Me3-TACN) note 2. Then, demineralized water was added to increase the volume of the solution to 21 mL. At this stage, the alginate content of the sample was 1.4% by weight.

[0376] The bottle was placed in a warm water bath (50 °C) for 60 minutes before being removed and cooled in an ice bath. Finally, the dynamic viscosity was determined using a Brookfield viscosity meter as described below.

[0377] Note 1: A 2 mM solution of [Mn2(pO)3(Me3-TACN)2](CH3COO)2 (see stock description 12 above) was mixed with an equivalent volume of the 2 mM ascorbic acid stock solution (stock solution 7). Upon contact with ascorbic acid, the catalyst changed from orange / red to red / purple.

[0378] Note 2: 0.5 mL of Me3-TACN binder (stock solution 10) and MnSO4.H2O (stock solution 8) were mixed, stirred, and used as a catalyst source to be added to the alginate solution.

[0379] Dynamic Viscosity Analysis

[0380] The dynamic viscosity of the samples was measured using a Brookfield HBDV-II cone / plate viscometer equipped with a CPE40 spindle. The viscometer was connected to a water bath to maintain the temperature at 25 °C. The apparatus was controlled via computer (external mode) using Rheocalc software.

[0381] Initially, the viscometer was zeroed without the spindle. Then, the CPE-40 spindle was installed and the clearance between the bottom of the cone and the top of the cup was set according to Brookfield recommendations.

[0382] 0.5 mL of the treated alginate solution were placed in the beaker of Petition 870220076624, dated 08 / 25 / 2022, page 72 / 115 69 / 99 viscometer using a disposable plastic syringe before the apparatus was closed and the test program started. This program was set up as follows:

[0383] Step 1: the initial rotation speed of the cone spindle was set to 150 RPM.

[0384] Step 2: the cone rotated at the stated speed for 30 seconds.

[0385] Step 3: A viscosity measurement was taken.

[0386] Step 4: the rotation speed increased by 10 RPM and steps 2 to 4 were repeated.

[0387] The program stopped after a viscosity measurement was performed at a rotational speed of 200 RPM.

[0388] Results

[0389] Experiment 1 Determination of the molecular weight parameters of alginate treated with [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 / ascorbic acid combined with H2O2.

[0390] A sodium alginate solution was treated at pH 10.5 with 10 mM H2O2 and different concentrations of the catalyst [Mn2(pO)3(Me3TACN)2](CH3COO)2 premixed with 1 molar equivalent of ascorbic acid. The treatment was carried out for 60 minutes at 50 °C; a complete description can be found in Procedure 1 above.

[0391] At the end of the reaction time, the dynamic and intrinsic viscosity values ​​were measured as explained above. The results are shown in Table 2. Table 2: Dynamic and intrinsic viscosities of alginate treated with 10 mM H2O2 and 0 and 10 pM [Mn2(pO)3(Me3-TACN)2](CH3COO)2 and equimolar amounts of ascorbic acid, pH 10.5; Treatment time of 60 minutes at 50 °C. Sample No. Description Dynamic Viscosity Intrinsic Viscosity Mw (kDa) Mn (kDa) Mw / Mn Petition 870220076624, dated 08 / 25 / 2022, page 73 / 115 70 / 99 @200RPM (mPa.s) (dL / g) 1 Untreated 97.1 10.3 496 130 3.8 2 Pure, without catalyst 91.6 8.7 421 110 3.8 3 Catalyst 0.5 μM 85.2 7.2 347 90 3.8 4 Catalyst 1 μM 67.6 5.6 267 69 3.9 5 Catalyst 2 μM 40.2 3.9 189 48 3.9 6 Catalyst 10 μM 6.0 1.4 68 17 4.0

[0392] The results presented in Table 2 show that treatment with [Mη2(μ-0)3(Mθ3-TΑ0N)2](0H30ΘΘ)2 premixed with ascorbic acid leads to a clear decrease in both the dynamic and intrinsic viscosity of the alginate solution. Since intrinsic viscosity is linked to the length of the polymer chains (Mw - 5acolumn), it can be concluded that treatment with a catalyst leads to the cleavage of the polymer chains, shortening the polymer and making it more soluble.

[0393] Experiment 2 pH-dependent depolymerization of alginate by [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 and H2O2

[0394] These experiments were performed following Procedure 2 described above. Alginate solutions were treated with 20 mM H2O2 at pH 9.0, 9.5, 10.0, 10.5 and 11.0 with 0 (blanks), [MnIV2^-O)3(Me3-TACN)2](CH3COO)2 5 μM and 10 μM before their dynamic viscosity was measured after a 60-minute reaction at 50 °C (see Table 3 below). Petition 870220076624, dated 08 / 25 / 2022, pp. 74 / 115 71 / 99 Table 3: Influence of pH and [Mn2(pO)3(Me3-TACN)2](CH3COO)2 level in solution on alginate depolymerization. Conditions: 20 mM H2O2, [Mn2(pO)3(Me3-TACN)2](CH3COO)2 0, 2.5, 5 and 10 pM, pH 9-11; 1 h reaction time at 50 °C. Dynamic viscosity (mPa.s) [Mn2(pO)3(Me3- TACN)2]2+ pH 9.0 pH 9.5 pH 10.0 pH 10.5 pH 11.0 0 110.3 109.5 109.2 107.5 103.5 2.5 pM 99.7 76.8 60.6 56.6 64.8 5 pM 78.1 41.3 27.2 25.6 40.4 10 pM 47.9 16.2 11.9 11.1 37.4

[0395] The data presented in Table 3 show that an effective loss of viscosity can be obtained using [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 at various pH values. Under the tested conditions, the ideal pH for alginate depolymerization is around pH 10, but significant viscosity losses are observed from pH 9.0 to pH 11.0 (upper and lower pH values ​​tested). It should be noted that very low catalyst concentrations are sufficient to cause significant viscosity losses: losses were observed after treatment with only 2.5 pM of the product.

[0396] Experiment 3 pH-dependent depolymerization of alginate by [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 / ascorbic acid and H2O2.

[0397] These tests were performed in the same manner as presented in Experiment 2 above, but the catalyst solution was premixed with a molar equivalent of neutralized ascorbic acid before being added to the alginate solution. The results are shown below. Table 4: Influence of pH and [Mn2(pO)3(Me3-TACN)2](CH3COO)2 level on the alginate depolymerization solution. Conditions: 20 mM H2O2, [Mn2(p Petition 870220076624, dated 08 / 25 / 2022, pp. 75 / 115 72 / 99 O)3(Me3-TACN)2](CH3COO)2 0, 2.5, 5 and 10 μM premixed with 1 molar equivalent of ascorbic acid at pH 9-11; 1h reaction time at 50 °C. Dynamic viscosity (mPa.s) [Mn2(pO)3(Me3-TACN)2]2+ / Ascorbic acid (1 / 1) pH 9.0 pH 9.5 pH 10.0 pH 10.5 pH 11.0 0 99.8 98.3 99.0 97.2 90.6 2.5 μM 50.1 33.8 31.3 41.5 53.2 5 μM 38.5 20.1 14.3 19.1 35.8 10 μM 20.3 10.5 6.9 10.1 32.3

[0398] The data in Table 4 show that premixing the catalyst with ascorbic acid before the reaction leads to a very pronounced loss of viscosity of the alginate, many times greater than when the catalyst is used without ascorbic acid (see Table 3). This is especially noticeable when using low levels of catalyst. The highest activity is observed over a wide pH range with an optimum pH of around 9.5-10.5.

[0399] Experiment 4 Alginate depolymerization dependent on [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 and H2O2 in carbonate buffer

[0400] The tests described in Experiment 2 were repeated with 4.7 mM hydrogenated sodium carbonate added to the alginate solution. The pH range was investigated from pH 8.0 to 10.5. The results are shown in Table 5. Table 5: Influence of pH and [Mn2^-O)3(Me3-TACN)2](CH3COO)2 level on the alginate depolymerization solution. Conditions: H2O2 20 mM; NaHCO3 4.7 mM; [Mn2^-O)3(Me3-TACN)2](CH3COO)2 0, 2.5, 5 and 10 μM, pH 8 - 10.5; 1h reaction time at 50 °C. Dynamic viscosity (mPa.s) Petition 870220076624, dated 08 / 25 / 2022, pp. 76 / 115 73 / 99 [Mn2(m- O)3(Me3- TACN)2]2+ pH 8.0 pH 8.5 pH 9.0 pH 9.5 pH 10.0 pH 10.5 0 99 99.2 98.6 99.2 99.4 96.7 2.5 mM 85 72.2 55.7 59.5 81.2 67.5 5mM 50.9 38.6 24.1 28 61 57 10mM 26.3 16.1 9.4 12.8 38.1 49.4

[0401] The data presented in Table 5 show that an effective viscosity loss can be obtained by using [MnIV2(pO)3(Me3TACN)2](CH3COO)2 at various pHs in a solution containing carbonate. The addition of carbonate to the alginate solution led to a decrease in the optimum pH, which was found to be around pH 9. It should be noted that even at the lower end of the pH ranges tested (pH 8 - 8.5) the catalyst showed good depolymerization activity.

[0402] Experiment 5 pH-dependent depolymerization of alginate by [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 / ascorbic acid and H2O2 in carbonate buffer.

[0403] Similar experiments, as described in Experiment 3, were conducted, but now in the presence of 4.7 mM hydrogenated sodium carbonate. The results of the dynamic viscosity measurements are shown in Table 6 below. Table 6: Influence of pH and [Mn2(pO)3(Me3-TACN)2](CH3COO)2 level on the alginate depolymerization solution. Conditions: 20 mM H2O2, 0, 2.5, 5 and 10 pM [Mn2(pO)3(Me3-TACN)2](CH3COO)2 premixed with 1 molar equivalent of ascorbic acid in 4.7 mM NaHCO3 at pH 8 - 10.5; 1 h of mixing time. Petition 870220076624, dated 08 / 25 / 2022, p. 77 / 115 74 / 99 reaction at 50 °C. Dynamic viscosity (mPa.s) [Mn2(qO)3(Me3TACN)2]2+ / Ascorbic acid (1 / 1) pH 8.0 pH 8.5 pH 9.0 pH 9.5 pH 10.0 pH 10.5 0 100.2 100.1 98.8 98.4 99.9 97.8 2.5 qM 66.6 46.7 31.0 42.6 75.3 80.6 5 qM 34.8 21.6 14.6 26.4 61.1 66.7 10 qM 14.8 9.8 6.9 12.8 44.9 50.6

[0404] The mixture of [Mn2(qO)3(Me3-TACN)2]2+ and ascorbic acid was very active in the depolymerization of alginate. As seen in Experiment 4, the addition of carbonate buffer leads to an increase in activity at low pH, with an optimum pH of about 9. When the catalyst is allowed to react with ascorbic acid before the alginate treatment process, the activity at pH 8.0-8.5 is clearly higher than when using the untreated catalyst solution.

[0405] Experiment 6 Temporal dependence of alginate depolymerization by [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 / ascorbic acid and H2O2 in carbonate buffer.

[0406] Tests similar to those described in Experiment 5 were performed at pH 8.5 and pH 9.5. Dynamic viscosity was determined after different reaction times ranging from 10 to 60 min. The results are presented in Table 7. Table 7: Temporal dependence of alginate depolymerization by [Mn2(qO)3(Me3-TACN)2](CH3COO)2 mixed with ascorbic acid. Conditions: H2O2 20 mM; NaHCO3 4.7 mM; pH 8.5 and pH 9.5; [Mn2(qO)3(Me3-TACN)2](CH3COO)2 0, 2.5, 5 and 10 qM premixed with 1 molar equivalent of ascorbic acid. Treatment times 10 min, 30 min and 1 h at 50 °C. Petition 870220076624, dated 08 / 25 / 2022, pp. 78 / 115 75 / 99 Dynamic viscosity (mPa.s) [Mn2(gO)3(Me3- TACN)2]2+ / Ascorbic acid (1 / 1) 10 min pH 9.5 30 min pH 9.5 60 min pH 9.5 10 min pH 8.5 30 min pH 8.5 60 min pH 8.5 0 99.8 100.7 97.8 99.8 98.1 92.6 2.5 gM 55.5 46.3 42.6 51.8 46.4 46.0 5 gM 28.5 24.7 19.9 24.6 26.0 21.1 10 gM 14.3 13.1 9.7 11.6 11.9 9.8

[0407] The data presented in Table 7 indicate that the depolymerization rate was very high under the tested conditions, with a large loss of viscosity measured after only 10 minutes of treatment.

[0408] Experiment 7 Reduction of alginate treatment temperature by [Mnrv2(pO)3(Me3-TACN)2](CH3COO)2 / ascorbic acid and H2O2 in carbonate buffer.

[0409] Experiments similar to those described in “

[0401] ” were carried out, but at 30 °C instead of 50 °C. The reaction time was also reduced to 30 minutes vs. 60 minutes previously.

[0410] Alginate solutions were treated at various pH values ​​using the catalyst pre-treated with ascorbic acid before the dynamic viscosity values ​​were measured. The results are presented in Table 8. Table 8: Alginate depolymerization by [Mn2(gO)3(Me3 Petition 870220076624, dated 08 / 25 / 2022, pp. 79 / 115 76 / 99 [TACN)2](CH3COO)2 mixed with ascorbic acid. Conditions: H2O2 20 mM; NaHCO3 4.7 mM; pH 8.0 to 10.0; [Mn2(pO)3(Me3-TACN)2](CH3COO)2 0, 2.5, 5 and 10 pM premixed with 1 molar equivalent of ascorbic acid; reaction time 30 minutes at 30 °C. Dynamic viscosity (mPa.s) [Mn2(pO)3(Me3TACN)2]2+ / Ascorbic acid (1 / 1) pH 8.0 pH 8.5 pH 9.0 pH 9.5 pH 10.0 0 99.3 100.5 97.8 98.6 95.4 2.5 pM 64.6 45.0 29.3 48.9 81.1 5 pM 30.6 20.4 13.0 29.0 68.8 10 pM 13.2 11.9 6.9 17.1 52.8

[0411] The data in Table 8 indicate that at 30 °C, the catalyst activity for alginate depolymerization is very high across a range of pH values.

[0412] Experiment 8 Alginate depolymerization by [MnlV2(pO)3(Me3TACN)2](CH3COO)2 / ascorbic acid with reduced levels of H2O2 in carbonate buffer.

[0413] The tests were carried out as described in “0” with a pH of 9 and an H2O2 concentration ranging from 0.5 to 10 mM. The measured dynamic viscosity values ​​of the solutions after treatment are presented in Table 9. Table 9: Dependence of [H2O2] on the depolymerization of alginate by [Mn2(pO)3(Me3-TACN)2](CH3COO)2, mixed with ascorbic acid. Conditions: H2O2 0.5-10 mM; NaHCO3 4.7 mM; pH 9.0; [Mn2(pO)3(Me3-TACN)2](CH3COO)2 0, 2.5, 5 and 10 pM premixed with 1 molar equivalent of ascorbic acid; Treatment time of 30 minutes at 30°C. Petition 870220076624, dated 08 / 25 / 2022, pp. 80-115 77 / 99 Dynamic viscosity (mPa.s) [Mn2(pO)3(Me3-TACN)2]2+ / Ascorbic acid (1 / 1) 0.5 H2O2 1.25 H2O2 2.5 H2O2 5.0 H2O2 10.0 H2O2 0 99.5 99.4 99.7 100.9 100.7 2.5 pM 45.7 25.8 27.6 20.4 22.3 5 pM 39.9 19.4 16.0 11.3 12.3 10 pM 39.8 17.8 10.7 12.3 7.5

[0414] The data gathered in Table 9 show that even when low levels of H2O2 are present, high alginate depolymerization activity is observed when the manganese catalyst is used.

[0415] Experiment 9 Alginate depolymerization by [MnlV2(pO)3(Me3TACN)2] (CH3COO)2 / various amounts of ascorbic acid and H2O2 in carbonate buffer.

[0416] Experiments similar to those described in “0” were carried out at pH 9.5 using various ratios of ascorbic acid: Mn catalyst. The results are presented in Table 10. Table 10: Alginate depolymerization by [Mn2(pO)3(Me3TACN)2](CH3COO)2 mixed with different molar ratios of ascorbic acid; Molar ratios of: [Catalyst] / [ascorbic acid] 1:0.5, 1:1, 1:2 and 1:3. Conditions: H2O2 20 mM; NaHCO3 4.7 mM; pH 9.5; [Mn2(pO)3(Me3TACN)2](CH3COO)2 0, 2.5, 5 and 10 pM premixed with various amounts of ascorbic acid; Treatment time 30 min at 30 °C. Dynamic viscosity (mPa.s) [Mn2(pO)3(Me3-TACN)2]2+ / Ascorbic acid 1:0.5 1:1 1:2 1:3 Petition 870220076624, dated 08 / 25 / 2022, pp. 81 / 115 78 / 99 0 97.7 97.7 97.7 97.7 2.5 qM 52.7 51.1 50.6 38.6 5 qM 32.8 26.5 24.7 22.6 10 qM 16.7 13.6 9.9 11.3

[0417] The data shown in Table 10 show that various molar ratios of ascorbic acid: catalyst can be used to achieve high alginate depolymerization activity.

[0418] Experiment 10 Alginate depolymerization by [MnIV2(pO)3(Me3TACN)2](CH3COO)2 / ascorbic acid with tBuOOH in carbonate buffer.

[0419] Experiments similar to those in “

[0401] ” were performed at pH 9.5, replacing H2O2 with tBuOOH. The results are presented in Table 11. Table 11: Alginate depolymerization by [Mn2(qO)3(Me3TACN)2](CH3COO)2 / ascorbic acid. Conditions: 20 mM tBuOOH; 4.7 mM NaHCO3; pH 9.5; [Mn2(qO)3(Me3-TACN)2](CH3COO)2 0, 2.5, 5 and 10 qM premixed with 1 molar equivalent of ascorbic acid; Treatment time 60 min at 50 °C. Dynamic viscosity (mPa.s) [Mn2(qO)3(Me3-TACN)2]2+ / Ascorbic acid pH 7.5 pH 8.0 pH 8.4 pH 9.0 0 100.5 101.6 97.8 nd 2.5 qM ndndnd 36.8 5 qM 40.4 27.6 24.6 nd 10 qM 22.2 14.1 14.4 nd and: not performed

[0420] The data in Table 11 show that effective depolymerization of alginate also occurs when using tBuOOH in combination with the catalyst of Petition 870220076624, dated 08 / 25 / 2022, p. 82 / 115 79 / 99 Mn at various pH levels.

[0421] Experiment 11 Alginate depolymerization by [MnIV2(pO)3(Me3TACN)2](CH3COO)2 / ascorbic acid with tBuOOH and H2O2 in carbonate buffer.

[0422] Experiments similar to those described above in Experiment 10 were performed using a mixture of tBuOOH and H2O2. The results are presented in Table 12. Table 12: Alginate depolymerization by [Mn2(pO)3(Me3TACN)2](CH3COO)2 / ascorbic acid. Conditions: 20 mM tBuOOH or 10 mM tBuOOH + 10 mM H2O2 or 20 mM H2O2. 4.7 mM NaHCO3; pH 8.5; 0, 2.5, 5 and 10 pM [Mn2(pO)3(Me3-TACN)2](CH3COO)2 premixed with 1 molar equivalent of ascorbic acid; Treatment time 60 min at 50 °C. Dynamic viscosity (mPa.s) [Mn2(pO)3(Me3-TACN)2]2+ / Ascorbic acid tBuOOH tBuOOH + H2O2 H2O2 0 101.6 100.5 97.2 2.5 pM 37.3 42.0 44.0 5 pM 22.5 22.4 21.2 10 pM 12.4 10.3 9.7

[0423] The data presented in Table 12 show that the activity of the solutions of the invention comprising tBuOOH mixed with H2O2 is very similar to that of the solutions of the invention comprising tBuOOH or H2O2 (when the total concentration of oxidant is kept equal).

[0424] Experiment 12 Alginate depolymerization by [MnIV2(pO)3(Me3TACN)2](CH3COO)2 / ascorbic acid with peracetic acid and H2O2 in carbonate buffer.

[0425] Experiments similar to those presented in “0” were carried out, Petition 870220076624, dated 08 / 25 / 2022, page 83 / 115 80 / 99 with peracetic acid (without hydrogen peroxide) or with a mixture of peracetic acid (PAA) and hydrogen peroxide (H2O2). The reaction time was extended from 30 to 60 minutes. The results are presented in Table 13. Table 13: Alginate depolymerization by [Mn2(pO)3(Me3TACN)2](CH3COO)2 / ascorbic acid. Conditions: 3 mM PAA or 1.5 mM PAA + 1.5 mM H2O2 or 3 mM H2O2, 0.185M Tris buffer; pH 8.5; 0 or 10 pM [Mn2(pO)3(Me3-TACN)2](CH3COO)2 or 10 pM [Mn2(pO)3(Me3TACN)2](CH3COO)2 premixed with 1 molar equivalent of ascorbic acid; Treatment time 60 min at 30 °C. Dynamic viscosity (mPa.s) Catalyst PAA 3 mM PAA 1.5 mM + H2O2 1.5 mM H2O2 3 mM None 102.8 98.8 104.9 [Mn2(pO)3(Me3-TACN)2]2+ 10 pM 94.8 85.3 49.4 [Mn2(pO)3(Me3-TACN)2]2+ 10 pM / Ascorbic acid 80.4 51.4 20.0

[0426] The data presented in Table 13 show that Mn catalysts can be used in combination with H2O2, peracetic acid, or a mixture of the two oxidants to depolymerize alginate. The catalyst activity without premixing ascorbic acid is much lower when PAA is present in solution (vs. H2O2 alone), but premixing the catalyst with ascorbic acid allows for a significant increase in the depolymerization of alginate chains.

[0427] Experiment 13 Alginate depolymerization by Mn-SO4 and Me3-TACN ligand with H2O2 in carbonate buffer.

[0428] These tests were carried out as described in “Experiment 5”, Petition 870220076624, dated 08 / 25 / 2022, pp. 84 / 115 81 / 99 using a mixture of MnSO4 and MeaTACN binder combined with hydrogen peroxide. The results are presented in Table 14. Table 14: Alginate depolymerization by [Mn2(pO)3(Me3TACN)2](CH3COO)2 vs. a combination of Mn salt and Me3TACN. Conditions: H2O2 10 mM, NaHCO3 4.7 mM; pH 9, 10 or 11; 0 or 5 pM [Mn2(pO)3(Me3-TACN)2](CH3COO)2 or Mn2+10 pM + Me3TACN 10 pM; treatment time 60 min at 50 °C Dynamic viscosity (mPa.s) Catalyst pH 9 pH 10 pH 11 None 95.5 95.5 95.5 [Mn2(pO)3(Me3-TACN)2]2+ 5 pM 21.6 40.6 62.1 (Mn2+ + Me3-TACN) 10 pM 61.9 69.9 71.6

[0429] The data presented in Table 14 show that both the pre-formed [Mn2(pO)3(Me3-TACN)2](CH3COO)2 complex and the combination of Mn2+ with Me3TACN ligand significantly reduce viscosity after treatment of alginate with H2O2.

[0430] Experiment 14 Alginate depolymerization by MnCl2.4H2O and Me3-TACN ligand in combination with tert-butyl hydroperoxide

[0431] Experiments similar to those described in “0” were performed using borate as buffer and tert-butyl hydroperoxide as oxidant. The results of these experiments are shown in Table 15. Table 15: Alginate depolymerization by [Mn2(pO)3(Me3TACN)2](CH3COO)2 vs. a combination of Mn chloride and Me3TACN. Conditions: 20 mM tBuOOH, 0.25 g / L sodium tetraborate decahydrate pH 9; or 5 pM [Mn2(pO)3(Me3-TACN)2](CH3COO)2 or 10 pM MnCl2 + 10 pM Me3TACN; treatment time 60 min at 50 °C Petition 870220076624, dated 08 / 25 / 2022, pp. 85 / 115 82 / 99 Dynamic viscosity (mPa.s) Catalyst pH 9 None 102.8 [Mn2(pO)3(Me3-TACN)2]2+ 5 pM 52.3 (Mn2+ + Me3-TACN) 10 pM 72.1

[0432] The results of the experiments presented in Table 15 show that a combination of Mn2+ and Me3TACN ligands is also active in the depolymerization of alginate when used with tBuOOH.

[0433] Experiment 15 Alginate depolymerization by a combination of catalyst, Me3TACN ligand and hydrogen peroxide.

[0434] These experiments were performed by treating a buffered carbonate solution (pH 10) containing 1.4% sodium alginate, 20 mM H2O2, Me3-TACN ligand; and MnSO4, [Mn2^-O)3(Me3-TACN)2]2+ or [Mn2(pO)3(Me3TACN)2]2+ mixed with an equivalent of ascorbic acid (see Procedure 2, Note 1). The Me3-TACN ligand was added to all solutions such that the molar ratio of catalyst to ligand was 1:1, 1:2, or 1:4. Note that two experiments (entry numbers 7 and 11 in Ta 16 below) were performed without any ligand so that the results could be used as references. Table 16: Alginate depolymerization by a combination of H2O2, catalyst and Me3TACN binder. Conditions: H2O2 20 mM, NaHCO3 4.7 mM; pH 10; MnSO4 5 μM or [Mn2(pO)3(Me3-TACN)2](CH3COO)2 2.5 pM - 5 pM or [Mn2(pO)3(Me3-TACN)2](CH3COO)2 2.5 pM premixed with 2.5 μM neutralized ascorbic acid, Me3-TACN binder 2.5-20 μM; treatment time 60 min at 50 °C. Inlet Catalyst Ascorbic acid Binder Me3TACN Dynamic viscosity (mPa.s) Petition 870220076624, dated 08 / 25 / 2022, p. 86 / 115 83 / 99 1 MnSO410 μΜ 10 85.4 2 MnSO410 μΜ 20 76.1 3 MnSO410 μΜ 40 63.4 4 [Mn2^-O)3(Me3-TACN)2]2 2.5 μΜ 2.5 2.5 72.5 5 [Mn2^-O)3(Me3- TACN)2]2+ 2.5 μΜ 2.5 5 69.3 6 [Mn2^-O)3(Me3- TACN)2]2+ 2.5 μΜ 2.5 10 61.7 7 [Mn2^-O)3(Me3- TACN)2]2+ 2.5 μΜ 78.1 8 [Mn2^-O)3(Me3- TACN)2]2+ 2.5 μΜ 2.5 73.3 9 [Mn2^-O)3(Me3- TACN)2]2+ 2.5 μΜ 5 66.6 10 [Mn2^-O)3(Me3- TACN)2]2+ 2.5 μΜ 10 56.8 11 [Mn2^-O)3(Me3- TACN)2]2+ 5 μΜ 61.0 12 [Mn2(pO)3(Me3- TACN)2]2+ 5 μΜ 5 54.0 13 [Mn2^-O)3(Me3- TACN)2]2+ 5 μΜ 10 47.7 14 [Mn2(pO)3(Me3- TACN)2]2+ 5 μΜ 20 38.1

[0435] The data presented in Table 16 show that the addition of the ligand Me3TACN to MnSO4, [Mn2^-O)3(Me3-TACN)2]2+ or [Mn2^-O)3(Me3-TACN)2]2+ mixed with an equivalent of ascorbic acid, leads in all cases to a significant decrease in the viscosity of the alginate, i.e., an increase Petition 870220076624, dated 08 / 25 / 2022, p. 87 / 115 84 / 99 of the depolymerization of alginate chains. Thus, adding Me3TACN binder to the manganese-containing Me3-TACN catalyst or adding molar excess of Me3-TACN binder to a manganese salt has a beneficial effect on system performance.

[0436] Experiment 16 Depolymerization of alginate with [Mn2(pO)2(pCH3COO)(Me4-DTNE)]Cl2, combined with H2O2

[0437] These experiments were performed by treating an alginate solution (1.4% in water) with 10 mM hydrogen peroxide and [Mn2(pO)2(pCH3COO)(Me4-DTNE)]2+. The pH ranged from pH 7.5 to pH 9 and no buffer was used. The catalyst level in solution was 0 μM (reference experiment), 2.5 μM or 5 μM. The results of the experiments are presented in Table 17. Table 17: Viscosity loss of an alginate solution after treatment with H2O2 and [Mn2(pO)2(p-CH3COO)(Me4-DTNE)]2+. Conditions: H2O2 10 mM, pH 7.5-9; [Mn2(pO)2(p-CH3COO)(Me4-DTNE)]2+ 0, 2.5 or 5 pM; treatment time 60 min. at 50 °C. Dynamic viscosity (mPa.s) Catalyst pH 7.5 pH 8.0 pH 8.5 pH 9.0 None 96.5 97.2 98.6 99.9 [Mn2(pO)2(p-CH3COO)(Me4-DTNE)]2+ 2.5 pM 82.3 82.9 80.7 nd [Mn2(pO)2(p-CH3COO)(Me4-DTNE)]2+ 5.0 pM 49.4 53.7 51.5 62.9 and: not performed

[0438] The data presented in Table 17 show that, under the tested conditions, the addition of [Mn2(pO)2(p-CH3COO)(Me4-DTNE)]2+ to the solutions leads to Petition 870220076624, dated 08 / 25 / 2022, pp. 88 / 115 85 / 99 a loss of viscosity. Furthermore, the viscosity of the alginate solutions treated with this catalyst was also reduced when the solution pH was low and close to neutral. These results are markedly different from those obtained when using [Mn2^-O)3(Me3-TACN)2]2+, where a greater loss of viscosity was observed at higher pHs (see Experiment 1).

[0439] Experiment 17 pH-dependent depolymerization of alginate by [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 and H2O2 in carbonate buffer.

[0440] The tests described in “Experiment 4” were repeated at 40 °C for one hour. The pH range investigated was from pH 8.0 to 10.5. The results are shown in Table 18. Table 18: Influence of pH and concentration of [Mn2(gO)3(Me3TACN)2](CH3COO)2 in solution on the degree of alginate depolymerization. Conditions: H2O2 20 mM; NaHCO3 4.7 mM; [Mn2(gO)3(Me3TACN)2](CH3COO)2 0, 5 and 10 gM, pH 8 - 10.5; reaction time of 1h at 40 °C. Dynamic viscosity (mPa.s) [Mn2(gO)3(Me3- TACN)2]2+ pH 8.0 PH 9.0 pH 9.5 PH 10.0 PH 10.5 0 94.9 92.0 91.0 94.7 91.6 5 gM nd 17.9 nd 54.2 nd 10 gM 24.2 9.1 10.5 nd 65.5 and: not performed

[0441] The data presented in Table 18 show that an effective viscosity loss can be obtained by using [MnIV2(pO)3(Me3TACN)2](CH3COO)2 at various pHs in a solution containing carbonate. It should be noted that, as in Experiment 4, a clear reduction in alginate viscosity was observed over a wide pH range.

[0442] Experiment 18 pH-dependent depolymerization of alginate by [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 / ascorbic acid and H2O2 in buffer of Petition 870220076624, dated 08 / 25 / 2022, pp. 89 / 115 86 / 99 carbonate.

[0443] The tests described in “Experiment 5” were repeated at 40 °C for one hour. The pH range investigated was from pH 8.0 to 10.5. The results are shown in Table 19. Table 19: Alginate depolymerization by [Mn2(pO)3(Me3TACN)2](CH3COO)2 premixed with ascorbic acid. Conditions: H2O2 20 mM; NaHCO3 4.7 mM; pH 8.0 to 10.5; [Mn2(pO)3(Me3-TACN)2](CH3COO)2 0, 5 and 10 pM premixed with 1 molar equivalent of ascorbic acid; 60 minutes reaction time at 40 °C. Dynamic viscosity (mPa.s) [Mn2(pO)3(Me3- TACN)2]2+ / Ascorbic acid (1 / 1) pH 8.0 pH 8.5 pH 9.0 pH 9.5 pH 10.0 pH 10.5 0 94.9 91.4 92.0 91.0 94.7 91.6 5 pM 32.8 23.5 12.6 18.5 56.3 67.6 10 pM 22.6 13.7 7.4 9.8 12.5 48.5

[0444] The data presented in Table 19 show that at various pHs, in a solution containing carbonate, an effective loss of viscosity can be obtained by using [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 combined with ascorbic acid. The highest activity is observed with a pH range of approximately pH 9-9.5. Again, as in Experiment 5, a clear reduction in alginate viscosity was observed over a wide pH range.

[0445] Experiment 19 Effect of the use of Dequest 2047 on the depolymerization of alginate by [MnIV2^-O)3(Me3] Petition 870220076624, dated 08 / 25 / 2022, p. 90 / 115 87 / 99 TACN)2](CH3COO)2 / ascorbic acid, H2O2 in carbonate buffer.

[0446] The tests described in “Experiment 18” were repeated using Dequest 2047 as a sequestrant, at 40 °C for one hour. The pH range investigated was pH 8.0 to 10.5. The results are shown in Table 20. Table 20: Alginate depolymerization by [Mn2(pO)3(Me3TACN)2](CH3COO)2 mixed with ascorbic acid. Conditions: H2O2 20 mM; NaHCO3 4.7 mM; Dequest 2047 0.1 mM; pH 8.0 to 10.5; [Mn2(pO)3(Me3TACN)2](CH3COO)2 0.10 pM premixed with 1 molar equivalent of ascorbic acid; 60 minutes reaction time at 40 °C. Dynamic viscosity (mPa.s) Dequest 2047 pH 8.0 pH 9.0 pH 10.0 pH 10.5 0 22.6 7.4 12.6 48.5 0.2 ndndnd 9.2 1 71.8 9.1 3.5 4.8 and: not performed

[0447] The data presented in Table 20 show the effect of adding Dequest 2047 to a carbonate-containing solution comprising hydrogen peroxide and different concentrations of [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 premixed with ascorbic acid. The data in Table 19 show that, in the absence of Dequest 2047, an effective viscosity loss is obtained at a pH of about 8.5 to 10. The data in Table 20 show that Dequest 2047 has a significant positive effect on alginate depolymerization at high pH (about 10 to 10.5), while at pH 8 there is a strong inhibitory effect of Dequest 2047 on alginate degradation activity. The viscosity loss increases at higher pHs when the amount of Dequest 2047 used increases from 0 to 1 mM.

[0448] Experiment 20 pH-dependent depolymerization of alginate by Petition 870220076624, dated 08 / 25 / 2022, pp. 91 / 115 88 / 99 [MnIV2(pO)3(Me3-TACN)2](PF6)2 / ascorbic acid and H2O2 in carbonate buffer.

[0449] Experiments similar to those in Experiment 18 were performed at 40 °C between pH values ​​of 8 to 10.5 using [MnIV2(pO)3(Me3-TACN)2](PF6)2 0, 5 and 10 μM premixed with ascorbic acid, and H2O2 in carbonate buffer. The results are presented in Table 21. Table 21: Alginate depolymerization by [MnIV2^-O)3(Me3-TACN)2](PF6)2 premixed with ascorbic acid. Conditions: H2O2 20 mM; NaHCO3 4.7 mM; pH 8.0 to 10.5; [MnIV2^-O)3(Me3-TACN)2](PF6)2 0, 5 and 10 μM premixed with 1 molar equivalent of ascorbic acid; reaction time 60 minutes at 40 °C. Dynamic viscosity (mPa.s) [MnIV2(pO)3(Me3TACN)2](PF6)2 / Ascorbic acid (1 / 1) pH 8.0 pH 8.5 pH 9.0 pH 9.5 pH 10.0 pH 10.5 0 94.9 91.4 92.0 91.0 94.7 91.6 5 pM 39.2 38.7 19.5 23.2 nd 65.1 10 pM 24.3 18.1 9.1 10.8 nd 49.0 and: not performed

[0450] The data presented in Table 21 show that in a solution containing carbonate at various pHs, an effective viscosity loss can be obtained by using [MnIV2^-O)3(Me3-TACN)2](PF6)2. The highest activity is observed with a pH range of approximately pH 8.5-9.

[0451] Comparison of the results in Tables 19 and 21 confirms that both catalyst salts (with acetate and PF6 counterions, respectively) show almost the same high activity in alginate depolymerization.

[0452] Experiment 21 Temperature-dependent depolymerization of alginate by [Mn2fr-O)3(Me3-TACN)2](CH3COO)2 / ascorbic acid and H2O2 Petition 870220076624, dated 08 / 25 / 2022, pp. 92-115 89 / 99 in carbonate buffer.

[0453] The optimal conditions for alginate depolymerization at 40 °C were repeated at room temperature for one hour. The results are shown in Table 22. Table 22: Alginate depolymerization by [Mn2(pO)3(Me3TACN)2](CH3COO)2 mixed with / without ascorbic acid. Conditions: H2O2 20 mM; NaHCO3 4.7 mM; Dequest 2047 0 or 1 mM; pH 9.0 to 10.5; [Mn2(pO)3(Me3-TACN)2](CH3COO)2 10 pM premixed with 0 or 1 molar equivalent of ascorbic acid; reaction time 60 minutes at room temperature. pH Dequest 2047(mM) Dynamic viscosity (mPa.s) [Mn2(pO)3(Me3-TACN)2]2+ / Ascorbic acid (1 / 1) 9.0 0 7.9 [Mn2(pO)3(Me3-TACN)2]2+ 9.5 0 19.3 [Mn2(pO)3(Me3-TACN)2]2+ / Ascorbic acid (1 / 1) 9.5 0 24.9 [Mn2(pO)3(Me3-TACN)2]2+ 10.5 1.0 42.6 [Mn2(pO)3(Me3-TACN)2]2+ / Ascorbic acid (1 / 1) 10.5 1.0 41.3 and: not performed

[0454] The data presented in Table 22 indicate the depolymerization of alginate at room temperature. The reference measurements (alginate treatment without Mn catalyst) gave dynamic viscosity values ​​between 90 and 100 mPa.s.

[0455] The data shown in Table 22 reveal that [Mn2(pO)3(Me3TACN)2](CH3COO)2 premixed with 0 or 1 molar equivalent of acid Petition 870220076624, dated 08 / 25 / 2022, pp. 93 / 115 90 / 99 ascorbic acid has a significant effect on alginate degradation at room temperature. In particular, when compared with the data presented in Table 19, experiments conducted at pH 9.0 show that a temperature reduction of approximately 20 °C leads to only a modest reduction in the degree of alginate degradation, with the final viscosity increasing from 7.4 (at 40 °C) to 7.9 (at room temperature).

[0456] Experiment 22 Removal of Pseudomonas aeruginosa biofilm by hydrogen peroxide mixtures (reference experiments)

[0457] This test was adapted from the 'ASTM standard design E2799 - 17 Standard test method for testing the efficiency of disinfection against Pseudomonas aeruginosa biofilm using the MBEC assay'. Pseudomonas aeruginosa bacterial inocula were prepared in Tryptone Soy Broth (TSB) to a cell density of 1 (+ / - 0.5) x 10⁶ CFU mL⁻¹. 150 pL of each bacterial inoculum were added to each well of two microtiter plates. Each Minimum Biofilm Eradication Concentration (MBEC) device was incubated at 37 °C and 110 rpm for 6 hours. After incubation, the established biofilms were rinsed three times in 200 pL of sterile distilled water in order to remove planktonic organisms. The rinsed biofilms were exposed to a challenge plate containing 200 pL of each H2O2 test mixture for one hour at 40 °C.The plates were then rinsed three times in 200 pL of sterile distilled water and then fixed by adding 300 pL of 95% ethanol for 15 min at room temperature (22-24 °C). The biofilm was stained with 150 pL of 0.1% Crystal Violet solution for 15 min at room temperature. The stained biofilms were rinsed three times with 200 pL of sterile distilled water to remove excess dye and were left to dry overnight. Then, 125 pL of 33% acetic acid was added to solubilize the Crystal Violet dye. The solubilized dye was then transferred to new, separate microtiter plates. Biofilm biomass was quantified by measuring optical density at 595 nm using a microtiter plate reader (Tecan Infinite Pro200). The tests were performed at... Petition 870220076624, dated 08 / 25 / 2022, pp. 94 / 115 91 / 99 triplicate.

[0458] The following checks were performed:

[0459] (1) Treat biofilms with phosphate buffer and 1% Tryptone Soy broth at 40 °C for one hour (negative control).

[0460] (2) Treatment of biofilms with hypochlorite solution (10%) at 40 °C for one hour (positive control).

[0461] The test results are as follows (all values ​​given are optical density at 595 nm from the solubilized crystal violet assay and provide a measure of the amount of biofilm remaining. A lower value means that more biofilm was removed due to the treatment).

[0462] The negative control provided an optical density of 0.56 (+ / - 0.06) - low biofilm removal - and the positive control an optical density of 0.07 (+ / - 0.01) - high biofilm removal. Table 23. Optical density of biofilms produced by Pseudomonas aeruginosa after one hour of treatment with the test mixtures. SD = Standard Deviation Standard. Conditions: 0.396 g / L sodium carbonate; pH 8.0 to 10.5; 0, 0.2 and _________________________1 mM Dequest 2047._________________________ H2O2 (mM) Dequest 2047 (mM) pH Optical density (OD595) (Mean ± SD) Mixture B1 20 0 8.0 0.23±0.05 Mixture B2 20 0 9.0 0.22±0.03 Mixture B3 20 0 10.5 0.30±0.04 Mixture B4 20 1.0 10.5 0.27±0.04 Mixture B5 20 0.2 10.5 0.26±0.03

[0463] The data presented in Table 23 show that solutions containing hydrogen peroxide removed the biofilm to give optical densities between 0.22 (mixture B2) and 0.30 (mixture B3). These results Petition 870220076624, dated 08 / 25 / 2022, pages 95 / 115 Studies 92 / 99 show that only moderate biofilm removal can be achieved by the different hydrogen peroxide solutions.

[0464] Experiment 23 Removal of Pseudomonas aeruginosa biofilm by [MnIV2fa-O)3(Me3-TACN)2](CH3COO)2 or [MnIV2&-O)3(Me3TACN)2](CH3COO)2 / ascorbic acid.

[0465] Carbonate buffer solutions (0.396 g / L sodium carbonate; pH 8 to 10.5), different levels of H2O2 (5, 10 and 20 mM), without and with Dequest 2047 (0.2 and 1 mM) with [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 5 or 10 pM or [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 - premixed with a molar equivalent of ascorbic acid -, were added to the microtiter plate with the biofilm as explained in Experiment 22. Table 24. Optical density of biofilms produced by Pseudomonas aeruginosa after one hour of treatment with the test mixtures. SD = Standard Deviation Standard. Conditions: 0.396 g / L sodium carbonate; pH 8.0 to 10.5; Dequest 2047 0, 0.2 and 1 mM; H2O2 20 mM. Dequest 2047 (mM) Mn Solution 1 * (pM) Mn Solution 2 * (pM) pH Optical Density (OD595) (Mean ± SD) Mixture 1 0 0 10.0 8.0 0.11±0.02 Mixture 2 0 0 10.0 9.0 0.12±0.02 Mixture 3 1.0 0 10.0 9.0 0.16±0.01 Mixture 4 0 0 10.0 9.5 0.14±0.01 Mixture 5 0 10.0 0 9.5 0.11±0.01 Mixture 6 1.0 5.0 0 10.0 0.16±0.03 Mixture 7 1.0 0 10.0 10.0 0.18±0.03 Mixture 8 0 10.0 0 10.0 0.11±0.01 Mixture 9 0 0 10.0 10.0 0.15±0.02 Petition 870220076624, dated 08 / 25 / 2022, pp. 96 / 115 93 / 99 Mixture 10 1.0 0 5.0 10.5 0.20±0.02 Mixture 11 1.0 5.0 0 10.5 0.21±0.01 Mixture 12 1.0 10.0 0 10.5 0.20±0.01 Mixture 13 0.2 0 5.0 10.5 0.17±0.01 *Solution of Mn 1 = [Mn2(μ-O)3(Mn3-TAON)2]2+*Solution of Mn 2 = [Mn2^-O)3(Me3-TACN)2]2+ / Ascorbic acid (1 / 1)

[0466] The data presented in Table 24 show that the optical densities at 595 nm of the Violet Dye ranged between 0.11 and 0.21 (with the negative control giving 0.46 and the positive control giving 0.12 in this experiment - see Experiment 22 for definitions of negative and positive to the control). Mixtures 1, 2, 5, and 8 give particularly low optical density values, showing that a large portion of the biofilm was removed. However, even the mixtures showing moderate biofilm removal (mixtures 10-13) are significantly better than the reference examples discussed in Experiment 22 (B3-B5).

[0467] The results of Experiments 22 and 23 clearly show that mixtures comprising low concentrations of [MnIV2(gO)a(Me3TACN)2](CH3COO)2 produce a significant improvement in biofilm removal compared to similar solutions that do not contain [MnIV2(gO)3(Me3-TACN)2](CH3COO)2. It was noted that the same conditions that led to a clear reduction in biofilm also led to a lower viscosity of alginate in experiments conducted using alginate as a model polysaccharide for biofilm (see above, Experiments 17-21).

[0468] Experiment 24 Removal of Staphylococcus epidermidis biofilm by hydrogen peroxide mixtures (reference experiments)

[0469] These experiments were performed in exactly the same manner as described for Experiment 22, except that now Staphylococcus epidermidis was used to generate the biofilm.

[0470] The negative control provided an optical density of 0.32 (+ / - 0.04) Petition 870220076624, dated 08 / 25 / 2022, pp. 97 / 115 94 / 99 - low biofilm removal - and the positive control an optical density of 0.06 (+ / - 0.00) - high biofilm removal. Table 25. Optical density of biofilms produced by Staphylococcus epidermidis after one hour of treatment with the test mixtures. SD = Standard Deviation. Conditions: 0.396 g / L sodium carbonate; pH 8.0 to 10.5; 0, 0.2 and Dequest 2047 at 1 mM. H2O2 (mM) Dequest 2047 (mM) pH Optical density (OD595) (Mean ± SD) Mixture B1 20 0.2 8.0 0.22±0.01 Mixture B2 20 0 9.0 0.19±0.01 Mixture B3 20 1.0 10.5 0.24±0.05

[0471] The data presented in Table 25 show that the application of hydrogen peroxide at different pHs leads to the removal of a moderate amount of biofilm (according to Experiment 22).

[0472] Experiment 25 Removal of biofilm ex Staphylococcus epidermidis by [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 or [MnIV2(pO)3(Me3TACN)2](CH3COO)2 / ascorbic acid.

[0473] These experiments were performed in exactly the same manner as described for Experiment 23, except that now Staphylococcus epidermidis was used to generate the biofilm. The conditions and results are shown in Table 26. Table 26. Optical density of biofilms produced by Staphylococcus epidermidis after one hour of treatment with the test mixtures. SD = Standard Deviation Standard. Conditions: 0.396 g / L of carbonate; pH 8.0 to 10.5; Dequest 2047 0, 0.2 and 1 mM; H2O2 20 mM. Petition 870220076624, dated 08 / 25 / 2022, pp. 98 / 115 95 / 99 Dequest 2047 (mM) Mn Solution 1 * (pM) Mn Solution 2 * (pM) pH Optical Density (OD595) (Mean ± SD) Mixture 1 0 0 10.0 8.0 0.09±0.02 Mixture 2 0 0 10.0 9.0 0.08±0.01 Mixture 3 1.0 0 10.0 9.0 0.09±0.01 Mixture 4 0 0 10.0 9.5 0.08±0.01 Mixture 5 0 10.0 0 9.5 0.07±0.01 Mixture 6 1.0 5.0 0 10.0 0.08±0.01 Mixture 7 1.0 0 10.0 10.0 0.09±0.01 Mixture 8 0 10.0 0 10.0 0.07±0.01 Mixture 9 0 0 10.0 10.0 0.17±0.03 Mixture 10 1.0 5.0 0 10.5 0.10±0.01 Mixture 11 1.0 10.0 0 10.5 0.09±0.02 Mixture 12 0.2 0 5.0 10.5 0.16±0.03 *Solution of Mn 1 = [Mn2(μ-O)3(Mn3-TAON)2]2+*Solution of Mn 2 = [Mn2^-O)3(Me3-TACN)2]2+ / Ascorbic acid (1 / 1)

[0474] The data presented in Table 26 show that treatment of biofilm produced by Staphylococcus epidermidis with many of the mixtures comprising [Mn2^-O)a(Me3-TACN)2]2+, with or without pretreatment with ascorbic acid, reduced the optical density of the biofilm between 0.07 and 0.1 (i.e., the treatment resulted in good biofilm removal). Mixtures 1-8, 10, and 11 in particular were highly active in biofilm removal. Although Mixtures 9 and 12 resulted in moderate levels of biofilm removal, these levels are substantially better than the negative control, which gave an optical density reading of 0.31.

[0475] Experiments 23 and 25 clearly show that the mixtures Petition 870220076624, dated 08 / 25 / 2022, pp. 99 / 115 96 / 99 comprising low concentrations of [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 produce a significant improvement in biofilm removal compared to reference solutions that do not contain [MnIV2(pO)3(Me3TACN)2](CH3COO)2.

[0476] The degradation of two biofilms originating from very different bacteria is clearly enhanced when [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 is present. Staphylococcus epidermidis are Gram-positive bacteria and contain murein. Pseudomonas aeruginosa are Gram-negative bacteria and contain alginate in EPS. The results described in Experiments 22-25 show that the enhanced biofilm degradation by aqueous mixtures comprising [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 is not restricted to biofilms comprising alginate.

[0477] Experiment 26 Removal of Pseudomonas aeruginosa biofilm in a CDC reactor by [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 or [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 / ascorbic acid

[0478] Based on the results of Experiments 23 and 25, additional tests were performed in a Center for Disease Control (CDC) biofilm reactor, which is a widely used standard configuration for studying biofilm formation and removal (for a recent review of various reactors for studying biofilms, see, for example, IB Gomes, et al., Critical Reviews in Biotechn., 38(5), 657-680 (2018).

[0479] This is a larger scale configuration that can be used to assess biofilm removal and count the number of bacteria after treatment. It is seen as a good model for realistic biofilm removal.

[0480] Pseudomonas aeruginosa cultured for 24 hours was harvested from a Tryptone Soy Agar (TSA) plate and used to prepare individual 1 (+ / - 0.5) x 10⁸ CFU mL⁻¹ suspensions in Tryptone Soy Broth (TSB). Each bacterial suspension was further diluted in TSB to prepare individual 1 (+ / - 0.5) x 10⁷ CFU mL⁻¹ suspensions. 400 mL of the bacterial inocula (the bacterial suspension) were transferred to separate sterile CDC reactors containing coupons of Petition 870220076624, dated 08 / 25 / 2022, pp. 100 / 115 97 / 99 polycarbonate (acquired from BIOSURFACE TECHNOLOGIES, diameter ~ 1.3 cm, thickness ~ 3.00 mm) on which biofilms are grown. Individual biofilms were grown for 24 h at 37 °C on a magnetic stirrer at 120 rpm. After 24 h of incubation, the pre-formed biofilms were washed with sterile distilled water to remove planktonic organisms. Pre-formed biofilms attached to the polycarbonate coupons were treated with mixtures comprising manganese complexes (described below) at 40 °C for 1 h. One set of treated coupons was analyzed using crystal violet staining (as described in Experiment 22) and a second set of coupons was analyzed to count the number of bacteria using the plate count method. All coupons were tested / analyzed in triplicate.

[0481] The first mixture contained 20 mM H2O2, without Dequest 2047, pH 9.0, 0.396 g / L of sodium carbonate buffer, [MnIV2(qO)3(Me3TACN)2](CH3COO)2 10 qM premixed with 1 molar equivalent of ascorbic acid (denoted below as 'first mixture').

[0482] The second mixture contained 20 mM H2O2, without Dequest 2047, pH 9.5, 0.396 g / L sodium carbonate buffer, 10 qM [MnIV2(qO)3(Me3TACN)2](CH3COO)2 (denoted below as 'second mixture').

[0483] The third mixture contained 20 mM H2O2, Dequest 2047 at 1 mM, pH 10.5, 0.396 g / L sodium carbonate buffer, 10 qM [MnIV2(qO)3(Me3TACN)2](CH3COO)2 (denoted below as 'third mixture').

[0484] A negative control was performed by treating the biofilms at room temperature for 1 h in phosphate buffer and TSB solution. A positive control was performed by treating the biofilms at 40 °C for 1 h, after which the coupons were sonicated and washed 3 times with distilled water.

[0485] The results of the experiments were as follows.

[0486] The negative control provided an optical density of 0.18 (+ / -0.11) and the positive control provided an optical density of 0.00 or a 100% reduction compared to the negative control. Petition 870220076624, dated 08 / 25 / 2022, pages 101 / 115 98 / 99

[0487] The first catalyst mixture provided a reading of 0.02 (+ / 0.01) or 89% reduction compared to the negative control.

[0488] The second catalyst mixture provided a reading of 0.05 (+ / 0.03) or 70% reduction compared to the negative control.

[0489] The third catalyst mixture provided a reading of 0.02 (+ / 0.03) or 87% reduction compared to the negative control.

[0490] Also, the biofilms were analyzed for various bacteria remaining after treatment. For this purpose, a recovery of viable Pseudomonas aeruginosa of 6.19 (+ / - 0.14) log CFUmL-1 was obtained from the negative control. No viable Pseudomonas aeruginosa was recovered after treatment with the positive control.

[0491] Analyses of biofilms treated with the first through third mixtures yielded the following Log bacterial counts and Log reduction in bacterial count compared to the negative control.

[0492] The first catalyst mixture provided a recovery of 4.57 (+ / -0.24) or a Log reduction of 1.63.

[0493] The second catalyst mixture provided a recovery of 4.91 (+ / -0.30) or a Log reduction of 1.28.

[0494] The third catalyst mixture provided a recovery of 3.04 (+ / -0.20) or a Log reduction of 3.15.

[0495] Experiment 27 Removal of biofilm ex Staphylococcus epidermis in a CDC reactor by [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 or [MnIV2(pO)3(Me3-TACN)2](CH3COO)2 / ascorbic acid

[0496] The same settings and procedures were followed as described in Experiment 26, except that, now, Staphylococcus epidermis biofilm was used in the CDC reactor.

[0497] The results of the experiments were as follows.

[0498] The negative control provided an optical density of 2.99 (+ / -0.64) and the positive control provided an optical density of 0.10 (+ / -0.64), which is a 96.5% reduction compared to the negative control. Petition 870220076624, dated 08 / 25 / 2022, pages 102 / 115 99 / 99

[0499] The first catalyst mixture provided a reading of 0.95 (+ / 0.34) or a 68% reduction compared to the negative control.

[0500] The second catalyst mixture provided a reading of 0.79 (+ / 0.13) or a 74% reduction compared to the negative control.

[0501] The third catalyst mixture provided a reading of 0.42 (+ / 0.18) or an 86% reduction compared to the negative control.

[0502] Also, the biofilms were analyzed for various bacteria remaining after treatment. For this purpose, a recovery of viable Staphylococcus epidermis of 7.29 (+ / - 0.36) log CFU mL-1 was obtained from the negative control. No viable Staphylococcus epidermis was recovered after treatment of the positive control.

[0503] Analyses of biofilms treated with the first to third mixtures produced the following Log bacterial counts and Log reduction in bacterial count compared to the negative control.

[0504] The first catalyst mixture provided a recovery of 6.02 (+ / -0.40) or a Log reduction of 1.27.

[0505] The second catalyst mixture provided a recovery of 6.44 (+ / -0.87) or a Log reduction of 0.85.

[0506] The third catalyst mixture provided a recovery of 5.12 (+ / -0.83) or a Log reduction of 2.17.

[0507] The results described in Experiments 26 and 27 clearly show that the first and third mixtures degrade the biofilm matrix produced by two different bacteria. These results are in complete agreement with the results discussed in Experiments 23 and 25. Furthermore, it was demonstrated that the number of residual bacteria was significantly lower after treatment with the first and third mixtures due to the partial removal of the biofilm. Petition 870220076624, dated 08 / 25 / 2022, pp. 103 / 115

Claims

1 / 3 Claims 1. A method for degrading a biofilm, characterized by comprising contacting the biofilm with an aqueous mixture comprising (i) a peroxide compound and (ii) a mononuclear manganese complex Mn(II), Mn(III) or Mn(IV), or binuclear Mn(II)Mn(II), Mn(III)Mn(II), Mn(III)Mn(III), Mn(III)Mn(IV) or Mn(IV)Mn(IV), wherein the aqueous mixture comprises a ligand of formula (I) or (II): (Q)p (I) R4R3C CR-ι R2 CR3R4 (II), wherein: --N-- [CRR2CR3R4) p is 3; each R is independently selected from the group consisting of hydrogen, C1-C24 alkyl, CH2Ce-C10 aryl, CH2CH2OH, CH2COOH, and pyridin-2-ylmethyl; Q' is an ethylene or propylene bridge; and R1, R2, R3, and R4 are independently selected from: H, C1-C4 alkyl, and C1-C4 hydroxyalkyl.

2. Method according to claim 1, characterized in that the ligand is 1,4,7-trimethyl-1,4,7-triazacyclononane or 1,2-bis(4,7-dimethyl-1,4,7-triazacyclonon-1-yl)-ethane.

3. A method according to claim 1 or claim 2, characterized in that the manganese complex comprises one or more non-coordination counterions selected from the group consisting of acetate, chloride, sulfate, nitrate, and hexafluorophosphate.

4. A method according to any of the preceding claims, characterized in that the manganese complex is any one or a combination selected from the group consisting of mononuclear complexes of Mn(II), Mn(III) and Mn(IV), and binuclear complexes of Mn(II)Mn(II), Mn(III)Mn(II), Mn(III)Mn(III) and Mn(III)Mn(IV).

5. Method according to any one of claims 1 to 3, characterized in that the manganese complex is a binuclear complex of Mn(III)Mn(III), Mn(III)Mn(IV) or Mn(IV)Mn(IV).

6. Method according to claim 1 or claim 2, characterized in that the manganese complex is part of a salt, wherein the salt is any one of the group consisting of [MnIVMnIV(pO)3(1,4,7-trimethyl-1,4,7-triazacyclononane)2][CH3COO]2, [MnIVMnIV(pO)3(1,4,7-trimethyl-1,4,7-triazacyclononane)2]2[SO4], [MnIVMnIV(pO)3(1,4,7-trimethyl-1,4,7-triazacyclononane)2][NO3]2, [MnIVMnIV(pO)3(1,4,7-trimethyl-1,4,7-triazacyclononane)2][PF6]2, or [MnIIIMnIV(pO)2(p-CH3COO)(1,2-bis(4,7-dimethyl1,4,7-triazacyclonon-1-yl)-ethane)][Cl]2.

7. Method, according to any of the preceding claims, characterized in that the molar ratio between the ligand of formula (I) and manganese is from 100:1 to 1.001:1 or in that the molar ratio between the ligand of formula (II) and manganese is from 50:1 to 0.5001:

1.

8. A method according to any of the preceding claims, characterized in that, prior to contact, a binuclear manganese compound Mn(III)Mn(III), Mn(III)Mn(IV) or Mn(IV)Mn(IV) comes into contact with a reducing agent, for example, ascorbic acid, to provide the manganese complex.

9. Method, according to claim 8, characterized by Petition 870220076624, dated 08 / 25 / 2022, pp. 105 / 115 3 / 3 manganese compound comprising a binder as defined in claim 1 or claim 2.

10. Method according to claim 8, characterized in that the manganese compound is [MnIVMnIV(pO)3(1,4,7-trimethyl-1,4,7-triazacyclononane)2][CH3COO]2, triazacyclononane)2]2[SO4], triazacyclononane)2][NO3]2, [MnIVMnIV(pO)3(1,4,7-trimethyl-1,4,7[MnIVMnIV(pO)3(1,4,7-trimethyl-1,4,7[MnIVMnIV(pO)3(1,4,7-trimethyl-1,4,7-triazacyclononane)2][PF6]2, or [MnIIIMnIV(pO)2(p-CH3COO)(1,2-bis(4,7-dimethyl-1,4,7-triazacyclononane)2] -yl)-ethane)][Cl]2.

11. A method for degrading a biofilm, characterized by comprising contacting the biofilm with an aqueous mixture comprising a peroxide compound and a binder as defined in claim 1 or claim 2.

12. A method according to any of the preceding claims, characterized in that the peroxide compound is any one or a combination of the group consisting of hydrogen peroxide, a peroxyacid, an alkyl hydroperoxide, and a phenyl alkyl hydroperoxide.

13. A method according to any one of claims 1 to 12, characterized in that the peroxide compound is a combination of hydrogen peroxide and peracetic acid.

14. A method, according to any of the preceding claims, characterized in that the pH of the aqueous mixture is from 6 to 12.

15. Method, according to any of the preceding claims, characterized by the biofilm comprising alginate. Petition 870220076624, dated 08 / 25 / 2022, pp. 106 / 115