Methods of ameliorating microbial growth

Abstract

A method of ameliorating microbial growth associated with equipment comprising the step of: contacting the microbial growth with an effective amount of a microbially active copolymer, wherein the microbially active copolymer comprises an acrolein derived segment and a polyalkylene glycol oligomer, and wherein the microbially active copolymer is obtainable by a process comprising reacting the polyalkylene glycol with acrolein in an aqueous solution to form the microbially active copolymer.

Description

Title of Invention[1] Methods of ameliorating microbial growthTechnical Field[2] This application claims priority from Australian Provisional Patent Application No. 2023904175 filed on 21 December 2023 the contents of which are to be taken as incorporated herein by this reference.[3] This invention generally relates to a method of ameliorating microbial growth associated with equipment, particularly equipment used in a gas field, an oil field, a commercial environment, marine environment or a naval environment, comprising the step of contacting the microbial growth an effective amount of a microbially active copolymer. The microbially active copolymer comprises an acrolein derived segment and a polyalkylene glycol oligomer. The microbially active copolymer is obtainable by a process comprising reacting the polyalkylene glycol with acrolein in aqueous solution to form the microbially active copolymer.Background of Invention[4] Biocides are generally employed in range of industries, in commerce and by consumers. Biocides are typically intended to control any harmful organism. Often biocides are employed in environments that are difficult access through physical and / or mechanical means. Often such environments include complex arrangements of equipment found in machinery and plumbing for example. This includes that found in gas and oil fields, engines, mines, naval settings and in heavy industry. Many consumer environments also suffer from harmful microbial growth, such as in, municipal water systems including housing piping and pool piping, medical environments, environments at risk of Legionnaires outbreaks, such as air conditioning (HVAC) systems and automotive equipment.[5] Currently existing biocides often employ commodity chemicals in preparations such as formulations of glutaraldehyde (1,5-pentanedial). Glutaraldehyde has been shown to be acutely toxic, particularly to aquatic life. During removal of microbial growth often significant quantities of this biocide and other commodity chemical-based biocides are released into the wider environment often causing significant ecological harm. [6] Other biologically active clinical entities such as small molecules and / or biologics are often not suitable as general-purpose biocides due to the prohibitive resources required in their manufacture. Several classes of small molecule antibiotics have also been shown to evince significant aquatic toxicity such as the phenylpropanoids, aminoglycosides as well as tetracycline antibiotics. Several classes of small molecule antifungal agents also a pose a serious harm to the aquatic environment such as the azole class of antifungals. [7] This is an example of the difficulties associated with the prediction of the toxic tolerance between classes of organisms. For example, the difference in the metabolism, physiology, and reproductive systems of mammalian versus marine organisms means that it is often not possible to predict whether one class of well-tolerated compounds in one set of organisms is likely to be well-tolerated in another. [8] Often the diaspora of microbial growth that causes issues in equipment, often having areas difficult to access through physical and / or mechanical means, is highly differentiated from those that cause infections in mammals such as humans. The hostile industrial and commercial environments as well as often anoxic environment leads to growth of microbes not often found in the mammalian environment. Such environments require biocidal treatment, as opposed to clinical management. Those environments often contain sulphur reducing bacteria (SRB), acid producing bacteria (APB), archaea, fungal species and those capable of forming microbial biofilms, including corrosion inducing biofilms. Due to the often highly differentiated metabolism and cellular operation of these species, it is not often possible to predict whether antimicrobials shown to act in the clinical mammalian environment would also be active on the microbial species that grow in industrial and / or commercial environments.[9] Biocides often need to be suitable for application into environments that are difficult to access through physical and / or mechanical means. In order to achieve this, and to minimise damage to equipment, desirable physicochemical characterises are required that allow application of the biocide as well as ensuring damage to sensitive equipment is minimised.

[10] The monetary value of biocides that can be used to ameliorate microbial growth associated with equipment in oil and gas fields alone has been estimated to be in the region of multibillion US dollars in 2022. It is difficult to quantify the ecological damage associated with release of significant amounts of currently used biocides.

[11] Accordingly, there is therefore an ongoing need for the development of alternative biocides that at least ameliorate at least one of the issues hereinbefore discussed.

[12] A reference herein to any matter is not to be taken as an admission that the matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.Summary of Invention

[13] The applicant has demonstrated that effective amounts of microbially active copolymers obtained from an acrolein derived segment and a polyalkylene glycol oligomer by reaction in aqueous solution can be used to ameliorate microbial growth associated with equipment. Microbially active copolymers representative of the claimed invention surprisingly show activity against microbes typically associated with microbial growth found in industrial and commercial settings such as SRB and APB. Microbially active copolymers representative of the claimed invention compare surprisingly well against commercially available biocides in terms of toleration of toxicity in tests simulating the aquatic environment. The microbially active copolymer has been examined for physiochemical properties and corrosiveness, the results of those tests indicate that microbially active copolymers representative of the invention are surprisingly suited to being used as a biocide in a method of ameliorating microbial growth. The copolymer may find use in environments where prevention of leakage of the microbially active material into the wider ecosystem, particularly the aquatic ecosystem, is difficult. Such environments include those where a gas field, oil field, commercial, marine or naval equipment is used.

[14] Accordingly, in one aspect of the invention there is provided a method of ameliorating microbial growth associated with equipment comprising the step of: contacting the microbial growth with an effective amount of a microbially active copolymer, wherein the microbially active copolymer comprises an acrolein derived segment and a polyalkylene glycol oligomer, and wherein the microbially active copolymer is obtainable by a process comprising reacting the polyalkylene glycol with the acrolein in an aqueous solution to form the microbially active copolymer.In another aspect of the invention there is provided a use of a microbially active copolymer to ameliorate microbial growth associated with equipment, wherein the microbial growth associated with equipment is contacted with an effective amount of the microbially active copolymer, wherein the microbially active copolymer comprises an acrolein derived segment and a polyalkylene glycol oligomer, and wherein the microbially active copolymer is obtainable by a process comprising reacting the polyalkylene glycol with the acrolein in aqueous solution to form the microbially active copolymer.

[15] In some embodiments the equipment is for use in a gas field, an oil field, a commercial environment, a marine environment or a naval environment.

[16] In some embodiments the method further comprises the step of at least in part discharging the microbially active copolymer from the equipment into an ecosystem following contacting the microbial growth with the effective amount of the microbially active copolymer. The applicant has surprisingly observed that the microbially active copolymer is well tolerated by aquatic organisms. Therefore, discharging of the microbially active copolymer can be conducted without substantially diluting the effective amount or detoxifying the microbially active copolymer directly or indirectly into ecosystems such as aquatic or aqueous ecosystems (such as the terrestrial water table) with reduced expectation of ecological harm seem in traditional biocides.

[17] In some embodiments the microbial growth is thermophilic or mesophilic. In further embodiments the microbial growth is halophilic, halotolerant or halophobic. In further embodiments the microbial growth is prone to cause corrosive damage. In further embodiments the microbial growth is sessile or vagile. In further embodiments the microbial growth is bacterial growth. In further embodiments the bacterial growth comprises acid producing bacteria or sulphur reducing bacteria.

[18] In some embodiments a stabilizer is present or added during the reacting the polyalkylene glycol with acrolein in aqueous solution to form a copolymer. In some embodiments a stabilizer is not present or added during the reaction.

[19] In some embodiments the stabilizer is an antioxidant. In some embodiments the antioxidant is hydroquinone. In some embodiments the hydroquinone is present in the range of about 0.1 to 0.5 wt% of the acrolein.

[20] In some embodiments the microbially active copolymer is formulated as a biocidal composition. In some embodiments the biocidal composition comprises unreacted free acrolein less than about 0.0001 %wt. In some embodiments the biocidal composition is aqueous. In some embodiments the aqueous biocidal composition has a water content is in the range of about 20 %wt to about 60 %wt water. In some embodiments the biocidal composition further comprising a gelling agent. In some embodiments the gelling agent is cellulose gum present in about 2 wt% or less. In some embodiments the microbially active copolymer is formulated with an additional stabiliser. In some embodiments the microbially active copolymer is not formulated with an additional stabiliser.

[21] In some embodiments the molecular weight of the polyalkylene glycol is in the range of about 100 to about 2500 Daltons. In some embodiments the molecular weight is about 200 Daltons.

[22] In some embodiments the polyalkyele glycol is polyethylene glycol. In some embodiments, the specific molecular weight of polyethylene glycol is about 190 to about 210.

[23] In some embodiments the molecular weight of the copolymer is about 200 Daltons to 7000 Daltons. In some embodiments the molecular weight of the copolymer is about 550 Daltons.

[24] In some embodiments the process is conducted at a temperature range of about -20 oC to about 50 oC. In some embodiments the pH of the solution is alkaline and no more than about pH 12.5.

[25] In some embodiments the effective amount is in the range of about 5 ppm to about 2000 ppm.

[26] In some embodiments contacting the microbial growth with an effective amount of a microbially active copolymer is conducted between about 60 seconds to 1 hour.

[27] In some embodiments the microbially active copolymer is thermally stable.

[28] In some embodiments the microbially active copolymer remains substantially non-formable having been subjected to agitation.

[29] In some embdiments the microbially active copolymer is substantially chemically inert to the equipment.

[30] In some embodiments the microbially active copolymer exhibits a corrosion rate of 1.00 mm / year or less at an effective amount of 1000 ppm as measured by linear polarization resistance.

[31] In some embodiments the microbially active copolymer exhibits a corrosion rate of about 0.5 mm / year or less at an amount of 10000 ppm as measured by immersion ASTM G1.

[32] In some embodiments the microbially active copolymer exhibits an EC50 for microalgal growth of 17.8 mg / mL over 78-hours and 61 ppm juvenile fish survival over 96-hours.

[33] In another aspect of the invention there is provided a kit for use in generating an aqueous biocidal composition copolymer comprising a microbially active copolymer for ameliorating microbial growth, wherein the kit comprises a polyalkylene glycol and acrolein, wherein reacting the polyalkylene glycol with acrolein in aqueous solution forms the aqueous biocidal composition comprising the microbially active copolymer. The reaction may be conducted using the conditions herein described.

[34] Further aspects of the invention appear below in the detailed description of the invention.Brief Description of Drawings

[35] Embodiments of the invention will herein be illustrated by way of example only with reference to the accompanying drawings in which:

[36] Figure 1 is a graph demonstrating that microbially active copolymers representative of the claimed invention are active against SRB and APB at a variety of amounts (in ppm) over the course of 1 hour (upper panel) and 24 hours (lower panel) simulated planktonic mesophilic freshwater conditions.

[37] Figure 2 is a graph demonstrating that microbially active copolymers representative of the claimed invention are active against SRB and APB at a variety of amounts (in ppm) over the course of 1 hour (upper panel) and 24 hours (lower panel) simulated planktonic thermophilic freshwater conditions.

[38] Figure 3 is a graph demonstrating that microbially active copolymers representative of the claimed invention are active against SRB and APB at a variety of amounts (in ppm) over the course of 1 hour (upper panel) and 24 hours (lower panel) simulated planktonic mesophilic brackish conditions.

[39] Figure 4 is a graph demonstrating that microbially active copolymers representative of the claimed invention are active against SRB and APB at a variety of amounts (ppm) over the course of 1 hour (upper panel) and 24 hours (lower panel) simulated planktonic thermophilic brackish conditions.

[40] Figure 5 is a graph demonstrating that microbially active copolymers representative of the claimed invention are active against SRB and APB at a variety of amounts (in ppm) over the course of 1 hour (upper panel) and 24 hours (lower panel) simulated sessile mesophilic freshwater conditions.

[41] Figure 6 is a graph demonstrating that microbially active copolymers representative of the claimed invention are active against SRB and APB at a variety of amounts (in ppm) over the course of 1 hour (upper panel) and 24 hours (lower panel) simulated sessile thermophilic freshwater conditions.

[42] Figure 7 is a graph demonstrating that microbially active copolymers representative of the claimed invention are active against SRB and APB at a variety of amounts (in ppm) over the course of 1 hour (upper panel) and 24 hours (lower panel) simulated sessile mesophilic brackish conditions.

[43] Figure 8 is a graph demonstrating that microbially active copolymers representative of the claimed invention are active against SRB and APB at a variety of amounts (in ppm) over the course of 1 hour (upper panel) and 24 hours (lower panel) simulated sessile thermophilic brackish conditions.

[44] Figure 9 is overlayed spectra of pre- and post- thermal aging of microbially active copolymers representative of the claimed invention showing that no significant chemical decomposition had occurred after treatment at 65 oC for 48 hours. Darker grey line is copolymers representative of the claimed invention before treatment, lighter line copolymers representative of the claimed invention after treatment.

[45] Figure 10 is a graph showing a stable base line achieved for microbially active copolymers representative of the claimed invention in a corrosivity test in CO2 enriched brine (1%NaCl) connected to API 5L X65 steel pipe, kept at 65 oC for 48 hours to simulate an annual corrosion rate.​

[46] Figure 11 is a chromatogram showing total ion concentration of a microbially active copolymer made from Good Manufacturing Practice (GMP) polyethylene glycol of average molecular weight 200 Daltons.

[47] Figure 12 is a chromatogram showing total ion concentration of a microbially active copolymer made from DOW Carobwax® polyethylene glycol of specific molecular weight of about 190 to about 210 Daltons.Definitions

[48] As used herein “equipment” refers to an inanimate item necessary for a particular purpose. Antimicrobial growth may be associated with a surface of the equipment and / or associated with media associated with the equipment, for example fluidic media such as liquids and / or gasses.

[49] As used herein “molecular weight” in Daltons refers to the number average molecular weight. Oligomers and copolymers may have species of specific molecular weights present outside of the average number molecular weight stated. As used herein “specific molecular weight” refers not to the average but the integer number in Daltons.

[50] As used herein “ameliorating” is to prevent, inhibit, delay, improve or remove the onset of one or more detectable observations of microbial growth or a complication thereof. For example, inhibit or delay development of the observation of damage and / or fouling to equipment caused by microbial growth.

[51] “Microbial growth” includes but is not limited to bacterial, viral, fungal, archaea and those capable of free movement (“vagile”) as well as those that are not and can form biofilms (“sessile”).

[52] An "effective amount" refers to at least an amount effective, at amounts and for periods of time necessary, to achieve the desired result. An effective amount can be provided in one or more applications. The effective amount may vary according to the desired result and according to the diaspora of microbial growth associated with the equipment. Typically, the effective amount will fall within a relatively broad range. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity. The microbial growth can be contacted with an effective amount in a single application or repeated once or several times over a period.

[53] The term “acrolein derived segment” refers to the copolymer segment comprising one or more acrolein monomer residues.

[54] The terms “oligomer”, “polyalkylene glycol oligomer” and “polyacrolein oligomer” refer to polymers consisting of at least two monomer units.

[55] The terms “monomer units” and “monomer residues” refer to units present in the copolymer derived from the reacting monomers such as acrolein and polyalkylene glycol.

[56] Where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all numerical values or sub-ranges in between these limits as if each numerical value and sub-range is explicitly recited. The statement "about X% to Y%" has the same meaning as "about X% to about Y%," unless indicated otherwise.

[57] The term “about” as used in the specification means approximately or nearly and in the context of a numerical value or range set forth herein is meant to encompass variations of + / - 10% or less, + / - 5% or less, + / - 1% or less, or + / - 0.1% or less of and from the numerical value or range recited or claimed.

[58] The term “parts per million (ppm)” when used in this specification in reference to microbial activity has been calculated based on an unrefined product of the reaction of polyalkylene glycol with acrolein in an aqueous solution.

[59] The term “substantially” as used in the specification means approximately or but for minor variations or within experimental measurement.

[60] Where the terms “comprise”, “comprises” and “comprising” are used in the specification (including the claims) they are to be interpreted as specifying the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof.

[61] Any subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.Detailed description

[62] The invention generally relates to a method of ameliorating microbial growth associated with equipment comprising the step of contacting the microbial growth an effective amount of a microbially active copolymer, wherein the microbially active copolymer comprises an acrolein derived segment and a polyalkylene glycol oligomer, and wherein the microbially active copolymer is obtainable by a process comprising reacting the polyalkylene glycol with acrolein in aqueous solution to form the microbially active copolymer.

[63] The invention also generally relates to uses and kits of the copolymer for microbial growth as herein described.

[64] In some embodiments, contacting of the effective amount of the microbially active copolymer to equipment requiring amelioration of microbial growth can be applied for example by spraying, pumping or application by hand.

[65] In some embodiments, the equipment used in gas fields or oil fields can include, for example the complex machinery used for storage, manipulation and movement of fluids. The equipment used in gas fields or oil fields can also include, for example structural support equipment. Such equipment includes, for example, drilling rigs, piping, drilling equipment, offshore and onshore rig equipment, solids and well control equipment, pumps, tanks, vacuum equipment, manifolds, valves, compressors, separators, gauges, engines, bearings, derricks, drives, casting heads, casting spools, tubing heads, distillation equipment, boilers, accumulators, heat exchangers, rig equipment and support equipment, hydraulic equipment and surfaces.

[66] In some embodiments, equipment in the commercial environment, can include for example municipal equipment, plumbing equipment, automotive equipment, aerospace equipment, aircraft equipment, surfaces, drains, heating equipment, refrigeration equipment medical environments, equipment exposed Legionnaires outbreaks, such as air conditioning (HVAC) systems and ovens.

[67] In some embodiments, equipment in a naval or marine environment can include, for example engines, boat hulls, bowsers, hydraulic equipment, marine infrastructures (including wharfs and piers for example), and transmission equipment.

[68] In some embodiments the method further comprises the step of at least in part discharging the microbially active copolymer from the equipment into an ecosystem following contacting the microbial growth with the effective amount of the microbially active copolymer. In some embodiments, discharging the microbially active copolymer from the equipment into the ecosystem is conducted without substantially diluting the effective amount or detoxifying the microbially active copolymer.

[69] Because the applicant has observed that the microbially active copolymer is well tolerated by aquatic animals, discharge of the copolymer may be conducted without significant dilution of the effective amount, detoxification and / or capturing and disposing of waster streams without causing ecological damage associated with current biocides such as Magnacide H for example. Thus reducing the resources used and associated with diluting the effective amount and / or capturing currently used biocides. In addition, the requirement of using various adsorbents, chemical detoxifying (bleaching for example) and / or physical detoxifying (for example burning) is expected to be reduced by the currently claimed method. Dilution of the effective amount can be up to a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000 or 10,000.

[70] Discharge can be direct, for example into aquatic ecosystems, such a marine ecosystems. Alternatively, it camnbe indirect, for example through sewage and / or other streams to enter terrestrial ecosystems and / or the water table. The discharge may be accidental caused by leakage in the equipment for example or non-accidental.

[71] In some embodiments the microbial growth is thermophilic. Thermophilic microbes prefer relatively high temperature environments, such as over 40 oC and in some cases survive up to 122 oC. Such temperatures can be found in equipment used in close proximity to man-made heat sources and / or due to geothermal heating. For example man-made heat sources include internal combustion, electrical heating, frictional heating and gas flares. Geothermal heating may be encountered by equipment used in mining and drilling operations.

[72] In some embodiments the microbial growth is mesophilic. Mesophilic microbes prefer relatively low temperature environments, such as below 40 oC. Such temperatures are encountered by equipment used during surface operations, shallow sub-surface operations and in the aquatic environment.

[73] In some embodiments the microbial growth is halophilic or halotolerant. Such microbes can tolerate the high-salinity found in brackish water or sea water. Equipment used in the high-salinity or brackish aquatic equipment that may be the subject of microbial growth includes as boat hulls, that found in oil rigs, that associated with fracking fluids, and some pools for example.

[74] In some embodiments the microbial growth is halotolerant. Such microbes prefer low-salinity aquatic environments found on land, lakes and rivers for example. Equipment used in the low-salinity aquatic equipment that may be the subject of microbial growth includes for example plumbing and fresh-water naval equipment.

[75] In some embodiments the microbial growth is sessile. Sessile microbial growth is incapable of independent movement and typically grows directly onto equipment. Often sessile microbial growth leads to the formation of biofilms. Biofilms are responsible for fouling and can lead to poor equipment performance and / or failure. Biofilms can also spread microbial growth throughout typically disparate environments, for example by attachment to the hulls of ships and marine infrastructures causing wider spread ecological harm. Microbially active copolymers representative of the current invention have been shown to be active against simulated sessile microbial growth. Infestation by sessile microbial growth can cause serious ecological and / or human harm, for example though toxic algal blooms.

[76] In some embodiments the microbial growth is vagile. Vagile microbial growth is capable of independent movement and / or typically is free to move in an aqueous environment. Such growth can damage equipment by blocking, clogging of equipment and can cause poor equipment performance and / or failure. Because such microbes can move, infestation by such microbes can be quickly established and widespread. Microbially active copolymers representative of the current invention have been shown to be active against simulated vagile microbial growth.

[77] In some embodiments the microbial growth may be prone to cause corrosive damage. Such microbes cause that cause damage to equipment include bacteria such as sulphur reducing bacteria (Desulphovibrio, Desulphotomaculum, and Desulphomonas sp.), iron reducing bacteria (Gallionellea ferrugine and Ferrobacillus sp.), acid producing bacteria (Pseudomonas, Aerobacter, and Bacillus), and sulphur oxidising bacteria (Thiobacillus sp.) for example.

[78] Corrosive damage can cause impaired operation and / or failure of equipment susceptible to corrosion. For example, equipment made of metals such as steel, iron, and aluminium alloys. Equipment made of concrete, glass and some plastics are also susceptible to microbial corrosion. Equipment used in sewerage, jet fuel and the petroleum industry is also susceptible to microbial corrosion.

[79] In some embodiments the bacterial growth comprises acid producing bacteria or sulphur reducing bacteria. Microbially active copolymers representative of the current invention have been shown to be active against acid producing bacteria and sulphur reducing bacteria found in oilfield wastewaters for example.

[80] Without wishing to be bound by theory, the diaspora of microbial growth found in the equipment used in environments hereinbefore described is often highly differentiated from that found in a clinical setting. An advantage observed by applicant is microbially active copolymers representative of the current invention are active in ameliorating microbes typical of microbial growth associated with these environments such as acid producing bacteria and sulphur reducing bacteria. The applicant has also shown that microbially active copolymers representative of the current invention are active in ameliorating microbial growth in both sessile and vagile simulations.

[81] The process used to obtain the microbially active copolymer comprises reacting the polyalkylene glycol with acrolein in aqueous solution to form the microbially active copolymer. Without wishing to be bound by theory, the process is amenable to large scale manufacture and is tolerant of a variety of conditions. Polyalkylene glycols and acrolein are available in large quantities as technical grade feed-stock chemicals.

[82] In some embodiments a stabilizer is present during the reacting the polyalkylene glycol with acrolein in aqueous solution to form a copolymer. In some embodiments a stabilizer is not present during the reaction. In some embodiments the stabilizer is an antioxidant. In some embodiments the antioxidant may be an optionally substituted hydroquinone, in some embodiments the antioxidant may be an organic antioxidant. In some embodiments the organic antioxidant may be an optionally substituted phenol such as 2,6-di-tert-butyl-4-methylphenol (BHT), in some embodiments the antioxidant may be ascorbic acid. In some embodiments the antioxidant may be an inorganic antioxidant. In some embodiments the inorganic antioxidant may be a sulphite such as a metabisulfite.

[83] In some embodiments the antioxidant is hydroquinone.

[84] In some embodiments antioxidant may be present in the range of about 0.1 to 5.0 wt% of the acrolein. In some embodiments antioxidant may be present in about 0.5%.

[85] In some embodiments hydroquinone may be present in the range of about 0.1 to 5.0 wt% of the acrolein. In some embodiments hydroquinone is present in about 0.5%.

[86] The applicant has shown that the process used to obtain the microbially active copolymer is tolerant to use of technical grade feed-stock chemicals used as received from chemical manufacturers without additional purification.

[87] In some embodiments the effective amount microbially active copolymer may be added to, mixed with and / or formulated with commercially available preparations such as paints, coatings, lubricants, hydraulics and / or efflux preparations before contacting the microbial growth associated with equipment.

[88] In some embodiments the microbially active copolymer is formulated as an biocidal composition. In some embodiments the microbially active copolymer is formulated as an aqueous biocidal composition. An aqueous composition can be readily manipulated by pumping, spraying or application by hand to equipment microbial growth associated with equipment. In some embodiments the formulation comprises about 20 to 60 wt% water. In some embodiments the formulation comprises about 30% to 50% water, in some embodiments the formulation comprises about 35 to 45% water, in some embodiments the formulation comprises about 40% water.

[89] In some embodiments the biocidal composition comprises unreacted free acrolein in an amount of about of less than 0.001% wt. In some embodiments the biocidal composition comprises unreacted free acrolein in an amount of about of less than 0.000.5% wt. In some embodiments the biocidal composition comprises unreacted free acrolein in an amount of about of less than 0.000.1% wt. In some embodiments the biocidal composition comprises unreacted free acrolein in an amount of about 0.001% to about 0.000.1% wt.

[90] In some embodiments the aqueous biocidal composition comprises a stabilizer. In some embodiments the aqueous biocidal composition does not comprises a stabilizer.

[91] In some embodiments the stabilizer is an antioxidant. In some embodiments the antioxidant is ascorbic acid, BHT (butylated hydroxytoluene), BHA (butylated hydroxyanisole), or other hydroquinone derivatives (for example mono-tertiary-butylhydroquinone).

[92] In some embodiments the antioxidant is present in the range of about 0.01 to about 0.5 wt% of the composition. In some embodiments the antioxidant can be present in the range of about 0.05 to about 0.4 wt% of the composition. In some embodiments the antioxidant can be present in the range of about 0.075% to about 0.3 wt% of the composition. In some embodiments the antioxidant can be present in the range of about 0.1% to about 0.1 wt% of the composition.

[93] In some embodiments, a gelling agent is used to increase the viscosity of the formulation. In some embodiments the gelling agent may be selected from agar, aliginate, carrageenan, carboxymethy cellulose, cellgulose gum, sodium pectate, gum tragacanth and mixtures thereof. In some embodiments the gelling agent may be present in the formation in about 1 to about 10 wt%. In some embodiments the gelling agent may be present in the formation in about 0.1 to about 10 wt%. In some embodiments the gelling agent may be present in the formation in about 0.1 to about 5wt%. In some embodiments the gelling agent may be present in the formation in about 0.5 to about 5wt%. In some embodiments the gelling agent may be present in the formation in about 1 to about 2wt%.

[94] In some embodiments the gelling agent is cellulose gum. In some embodiments, cellulose gum may be present in about 5 wt% or less. In some embodiments the cellulose gum may be present in about 4 wt% or less. In some embodiments the cellulose gum may be present in about 3 wt% or less. In some embodiments the cellulose gum may be present in about 2 wt% or less.

[95] Gelled formulations allow application of the microbially active copolymer to adhere more readily to surfaces of equipment to allow sufficient contact for amelioration of microbial growth. For example, formulations having increased viscosity may be applied as a paint and / or coating.

[96] In some embodiments, the acrolein derived segment may comprise one or more acrolein monomer residues. In one embodiment the acrolein derived segment comprises a polyacrolein oligomer.

[97] The polyalkylene glycol may be a poly(C1 to C4 alkylene glycol) or mixture or copolymer thereof but in general the polyalkylene glycol.

[98] In some embodiments the molecular weight of the polyalkylene glycol is about 100 Daltons to about 2500 Daltons. In some embodiments the molecular weight of the polyalkylene glycol is about 200 Daltons to about 2000. In some embodiments the molecular weight of the polyalkylene glycol is about 200 Daltons to about 1500. In some embodiments the molecular weight of the polyalkylene glycol is about 200 Daltons to about 1000. In some embodiments the molecular weight of the polyalkylene glycol is about 200 Daltons to about 800. In some embodiments the molecular weight of the polyalkylene glycol is about 200 Daltons to about 600.

[99] In some embodiments, the polyalkylene glycol is polyethylene glycol. Polyethylene glycol is of formula H--[OCH2CH2]n--OH. The average value of n is at least 3 and is generally from 3 to 13 (although the average need not be an integer). Polyethylene glycol is widely available from commercial suppliers in technical grade and is sold in specified nominal molecular weights which generally signify that the average molecular weight as discussed herein. The viscosities and methods for molecular weight determination are disclosed in USP NF Official Compendium of Standards Volume 11180-1182 [2007 Edition]. In one set of embodiments the polyethylene glycol is of molecular weight from 200 to 400. In some embodiments it may be preferred to use a specific oligomer of ethylene glycol such as the compound of formula H--[OCH2CH2]n--OH where n is 3 or 4.

[100] In the case of polyethylene glycol of average molecular weight 200, the upper and lower limits of specific molecular weight of specific species of about 100 Daltons and 460 Daltons respectively have been detected by mass-spectrometry.

[101] In some embodiments the molecular weight of the polyethylene glycol is about 100 Daltons to about 2500 Daltons. In some embodiments the molecular weight of the polyethylene glycol is about 200 Daltons to about 2000. In some embodiments the molecular weight of the polyethylene glycol is about 200 Daltons to about 1500. In some embodiments the molecular weight of the polyethylene glycol is about 200 Daltons to about 1000. In some embodiments the molecular weight of the polyethylene glycol is about 200 Daltons to about 800 Daltons.

[102] In some embodiments, the specific molecular weight of polyethylene glycol is between about 190 to about 210.

[103] In one set of embodiments the molecular weight of the copolymer is about 200 Daltons to about 7000 Daltons. In one set of embodiments the molecular weight of the copolymer is about 300 Daltons to about 5000 Daltons. In one set of embodiments the molecular weight of the copolymer is about 400 to about 4000 Daltons. In one set of embodiments the molecular weight of the copolymer is about 500 to about 200 Daltons. In one set of embodiments the molecular weight of the copolymer is about 800 Daltons to about 1000 Daltons. In some embodiments the copolymer is about 250 Daltons to about 800 Daltons. In some embodiments the copolymer is about 400 Daltons to about 800 Daltons.

[104] In some embodiments, the molecular weight may be limited to about 1000 Daltons or lower by controlling the ratio of monomers, the dilution of acrolein and polyethylene glycol with water keeping the pH in a lower range, maintaining the pH at no more than 12.0 and within a pH range of 12.0 to 7.0.

[105] In one set of embodiments the process for obtaining the microbially active copolymer comprises base catalyzed polymerization of acrolein in an aqueous solution comprising polyethylene glycol wherein the ratio of polyalkylene glycol / acrolein is at least 4, preferably at least 8, more preferably at least 10, and water is present in an amount of at least 20% by weight of the biocidal composition.

[106] In a preferred set of embodiments, the process comprises adding an aqueous solution of acrolein, preferably having an acrolein concentration of no more than 50%w / w, to an aqueous solution of polyethylene glycol comprising at least 10% w / w water and having a pH of no more than 14.0, preferably no more than pH 12.

[107] In a still more preferred embodiment acrolein is added as an aqueous solution to an aqueous solution of polyalkylene glycol of pH 9 to 12.

[108] In addition to acrolein monomer, other monomers e.g., acrylic acid, acrylamide, acrylonitrile, vinyl chloride, styrene, methacrylic acid, methyl methacrylate, vinyl acetate, vinyl pyridine and vinyl pyrrolidone may be used as additional monomers in preparation of copolymers comprising a polyalkylene glycol oligomer segment and acrolein derived segment, as described herein. The ratio of monomers may be chosen so as to maintain the water solubility of the copolymer and incorporation of other monomers may be controlled by reaction conditions and relative monomer concentrations bearing in mind monomer reactivity. In general it is preferred that other monomers constitute no more than 15 mole % of the monomer residues of the copolymer, preferably no more than 10 mole % and most preferable the copolymer only consists of polyalkylene glycol and acrolein monomer residues.

[109] In a preferred set of embodiments the process of obtaining the microbially active copolymer of the present invention comprises the following steps: providing a mildly basic (preferably of pH no more than 14.0; more preferably of pH 9 to 12) aqueous solution of a polyalkylene glycol (preferably polyethylene glycol of molecular weight in the range of from 200 to 600 Daltons); stirring the mildly basic solution vigorously to entrain air; adding (preferably slowly over a period such as at least 2 minutes, more preferably at least 5 minutes) acrolein as an aqueous solution of concentration no more than 50 % w / w of the acrolein aqueous solution (usually containing preservative); maintaining the reaction temperature in the range of from -20°C to 50°C; and once the acrolein monomer has been consumed, adding acid to provide a pH less than 9 and preferably no more than 8. Preferably no more than pH 7.5.

[110] The molecular weight of the resulting the microbially active copolymer is controlled by the molecular weight of the polyalkylene glycol, as well as being directly proportional to its hydroxyl concentration. The polymerization is exothermic and often requires temperature control, for example by use of a heat exchanger.

[111] As the reaction is exothermic, the process preferably includes removing heat of reaction from the zone of reaction of the acrolein monomer and polyalkylene glycol. Heat removal can be achieved by adding additional solvent, such as additional water, or reactants into the reaction zone or by transferring heat from the reaction zone via a commercially available and well-known indirect heat exchange device such as a shell and tube or spiral wound heat exchanger provided with a flow of a cooling fluid such as water.

[112] In one embodiment the effect of the exothermic reaction of acrolein and polyalkylene glycol may be controlled and the temperature maintained in the required range during preparation of the copolymer by carrying out the polymerisation reaction in a reaction vessel provided with indirect heat exchange to maintain the required temperature. In one embodiment the reaction vessel is a jacketed reaction vessel and indirect heat exchange is provided by flow of cooling fluid such as water in the jacket.

[113] In a preferred embodiment the heat of polymerisation may be removed from the reactor using indirect exchange with a cooling medium, such as water or other coolant fluid depending on the required temperature, in a jacket surrounding at least part of the reactor. The efficiency of heat removal may be computer controlled to maintain the temperature within the required range during the formation of the copolymer. The reactor may be a batch reactor having a jacket for flow of a cooling fluid, or may be a tubular reactor such as a tubular loop reactor comprising one or more jackets for the cooling fluid, such as water, concentrically surrounding at least part of the tubular reactor.

[114] Typically, the reaction vessel will include an agitator such as a stirrer or other means providing mixing to minimise the occurrence of relatively hot or cold regions as a result of the reaction exotherm and / or heat exchange.

[115] The reaction to form the copolymer may in one set of embodiments be carried out with a temperature in the range of about -20oC to 50 oC. The reaction to form the copolymer may in one set of embodiments be carried out with a temperature in the range of about -10oC to 40 oC. The reaction to form the copolymer may in one set of embodiments be carried out with a temperature in the range of about 0oC to 30 oC. The reaction to form the copolymer may in one set of embodiments be carried out with a temperature in the range of about 5oC to 20 oC. The reaction to form the copolymer may in one set of embodiments be carried out with a temperature in the range of about 0 oC to 10 oC. The reaction to form the copolymer may in one set of embodiments be carried out with a temperature in the range of about no more than 20 oC. The reaction to form the copolymer may in one set of embodiments be carried out with a temperature in the range of about no more than 10 oC. During the reaction the stirring is preferably continued, and the pH maintained mildly basic (preferably of pH no more than 12.0, more preferably of pH 9 to 11), only as necessary. The addition of more base and its concentration is minimized so as to lower degradation / side-reactions and to reduce carbonyl or carboxyl formation in the product.

[116] Finally, the pH of the solution may be buffered. In a preferred set of embodiments, the pH is adjusted to near neutral, by the addition of acid. The extremely pungent smell of acrolein is no longer evident in the copolymer product, which is formed in at least 99% yield.

[117] In some embodiments, the weight-ratio of acrolein: polyethylene glycol used in its preparation of the copolymer is preferably between 1:4 and 1:40, and more preferably about 1:8 to about 1:20.

[118] In some embodiments, the preferred base is an aqueous solution of an alkali hydroxide; more preferably, the alkali hydroxide is sodium hydroxide.

[119] The preferred acid is dilute hydrochloric acid and acetic acid is useful for pH buffering purposes. When using technical grade feed-stock chemicals, additional buffering is required.

[120] It is preferred that the addition of acrolein to the aqueous solution of polyalkylene glycol takes about 10 minutes for the reaction to reach completion, and the addition of acid generally takes place in about 10 to about 50 minutes, and preferably is no more than 90 minutes.

[121] Typically, the applicant has found that a reaction time of about 50 minutes is suitable to obtain virtually complete conversion to the copolymer product. The acrolein is preferably added to the aqueous polyalkylene glycol as an aqueous solution - more preferably as a concentration in the range of from about 10% to about 30% by weight of acrolein monomer, based on the weight of the aqueous acrolein solution to be added to the aqueous polyalkylene glycol solution.

[122] The effective amount is such that microbial growth associated with equipment can be ameliorated without requiring unfeasible amounts of the microbially active copolymer. The applicant has shown that the effective amount is comparable to commercially available biocides.

[123] In some embodiments the effective amount of the microbially active copolymer may be in the range of about 5 ppm to about 2000 ppm. In some embodiments the effective amount of the microbially active copolymer may be in the range of about 10 ppm to about 1900 ppm. In some embodiments the effective amount of the microbially active copolymer may be in the range of about 20 ppm to about 1800 ppm. In some embodiments the effective amount of the microbially active copolymer may be in the range of about 30 ppm to about 1700 ppm. In some embodiments the effective amount of the microbially active copolymer may be in the range of about 40 ppm to about 1600 ppm. In some embodiments the effective amount of the microbially active copolymer may be in the range of about 50 ppm to about 800 ppm. In some embodiments the effective amount of the microbially active copolymer may be in the range of about 50 ppm to about 400 ppm.

[124] This In some embodiments the effective amount for vagile APB may be about 10 ppm to 100 ppm. In some embodiments the effective amount for vagile APB may be about 20 ppm to 80 ppm.

[125] In some embodiments the effective amount for vagile APB may be about 10 ppm to 100 ppm. In some embodiments the effective amount for vagile APB may be about 20 ppm to 80 ppm. In some embodiments the effective amount for vagile APB may be about 30 ppm to 60 ppm. In some embodiments the effective amount for vagile APB may be about 50 ppm.

[126] In some embodiments the effective amount for vagile SRB may be about 600 ppm to 1000 ppm. In some embodiments the effective amount for vagile SRB may be about 700 ppm to 900 ppm. In some embodiments the effective amount for vagile SRB may be about 800 ppm.

[127] In some embodiments the effective amount for sessile APB may be about 10 ppm to 100 ppm. In some embodiments the effective amount for sessile APB may be about 20 ppm to 80 ppm. In some embodiments the effective amount for sessile APB may be about 30 ppm to 60 ppm. In some embodiments the effective amount for sessile APB may be about 50 ppm.

[128] In some embodiments the effective amount for sessile SRB may be about 50 ppm to 400 ppm. In some embodiments the effective amount for sessile SRB may be about 100 ppm to 300 ppm. In some embodiments the effective amount for sessile SRB may be about 200 ppm.

[129] The applicant has surprisingly observed that microbially active copolymers representative of the claimed invention are active in a wide variety of environments. These include vagile, sessile, thermophilic, mesophilic, brackish, freshwater and combination as thereof as herein described. The applicant has surprisingly observed that microbially active copolymers are particularly active against SRB and APB in such environments. In addition, the applicant has surprisingly observed that microbially active copolymers representative of the claimed invention are particularly active in challenging environments such as thermophilic, sessile, brackish and mixtures thereof.

[130] Without wishing to be bound by theory, relatively high salinity affects the osmotic stress on bacterial colonies. In such environments, the osmotic stress may reduce the uptake of the copolymers or otherwise likely reduce the efficacy of the copolymer. For similar reasons a reduction in efficacy may be expected for sessile and / or thermophilic colonies. Microbially active copolymers representative of the claimed invention performed well in all conditions observed.

[131] In some embodiments, the contacting time is about 60 seconds to 24 hours. In some embodiments, the contacting time is about an hour. This contact time is convenient to reduce the economic loss associated with the down-time required for removal of microbial growth. In some embodiments the contacting time is less than 10 minutes.

[132] In some embodiments, the contacting time for APB is less than about 1 hour. In some embodiments, the contacting time for APB is between about 1 minute to 30 minutes. In some embodiments, the contacting time for APB is between about 5 minutes to 10 minutes. In some embodiments, the contacting time for APB is between less than to 10 minutes.

[133] In some embodiments, the contacting time for SRB is about 1 hour to 24 hours. In some embodiments, the contacting time for SRB is between about 5 hours to 12 hours. In some embodiments, the contacting time for SRB is between about 8 hours and 10 hours.

[134] In some embodiments the microbially active copolymer is stable as measured by FTIR (Fourier-Transform Infrared Spectroscopy) after heating at 65 oC for 48 hours. Thus demonstrating that the copolymer is suitable to be applied in equipment that is above room temperature for prolonged periods of time without substantial degradation.

[135] In some embodiments, the microbially active copolymer remains non-formable having been subjected to vigorous agitation (such as shaking in a sealed tube for 1 hour). In some embodiments, the microbially active copolymer remains non-formable having been subjected to sparging with nitrogen gas for 10 minutes with 0.1 % sodium chloride solution or 50% low aromatic white spirit. In some embodiments, the microbially active copolymer remains non-formable and remains substantially the same volume after sparging with carbon dioxide at 80 oC for 1 hour. This demonstrates that the copolymer can be subject to significant agitation, such as that caused during shaking, stirring and pumping for example without causing substantial foaming.

[136] The applicant has shown that the microbially active copolymer has favourable physicochemical properties making it amenable to use in microbial growth associated with equipment typically used in the environments hereinbefore described. For example, lack of foaming and lack of sediment formation means that the microbially active copolymer is suitable for spraying, pumping and application by hand. The microbially active copolymer is also suitable to pumping into the complex equipment hereinbefore described.

[137] In some embodiments, the microbially active copolymer is substantially chemically inert to the equipment.

[138] In some embodiments, the microbially active copolymer copolymer exhibits a corrosion rate of 0.91 mm / year or less at an amount of 1000 ppm as measured by linear polarization resistance. In some embodiments, the microbially active copolymer exhibits a corrosion rate of about 0.5 mm / year or less at an amount of 10000 ppm as measured by immersion ASTM G1.

[139] Thus, demonstrating that the copolymer is well tolerated by equipment, especially made by metals that can be subject to corrosive action, such as iron and alloys thereof such as steel. Thus, in some embodiments, contacting the equipment with an effective amount the microbially active copolymer causes substantially no corrosion.

[140] In some embodiments the corrosion rate corrosion rate is about 0.01 mm / year to about 1.0 mm / year. In some embodiments the corrosion rate corrosion rate is about 0.02 mm / year to about 0.8 mm / year to. In some embodiments the corrosion rate corrosion rate is about 0.03 mm / year to about 0.6 mm / year. In some embodiments the corrosion rate corrosion rate is about 0.01 mm / year to about 0.5 mm / year. In some embodiments the corrosion rate corrosion rate is about 0.1 mm / year to about 0.5 mm / year.In some embodiments, the microbially active copolymer exhibits an EC50 (%) for microalgal growth of 17.8, over 78-hours and copepod survival of 7.63 over 48-hours or 61.0 juvenile fish survival over 96-hours.

[141] Without wishing to be bound by theory, such well-tolerated toxicity in the aquatic environment cannot be expected from well-tolerated mammalian toxicity due to the distinction in the metabolism, physiology, and reproductive systems of mammalian versus marine organisms. The applicant has demonstrated that microbially active copolymers representative of the present invention perform favourably compared to commercially available commodity chemical-based biocides such as glutaraldehyde. Thus the microbially active copolymers are suitable for discharge into an ecosystem without significant dilution of the effective amount and / or detoxifying.

[142] A further aspect of the invention is a kit for use in generating an aqueous biocidal composition copolymer comprising a microbially active copolymer for ameliorating microbial growth associated with equipment, wherein the kit comprises a polyalkylene glycol and acrolein, wherein reacting the polyalkylene glycol with acrolein in aqueous solution forms the biocide comprising the microbially active copolymer. The reaction of polyalkylene glycol with acrolein in aqueous solution can be performed according to the conditions herein disclosed.

[143] In some embodiments, the copolymer may be prepared in situ to ameliorate microbial growth associated with equipmentby reacting the polyalkylene glycol with acrolein in aqueous solution forms the biocide comprising the microbially active copolymer. The reaction of polyalkylene glycol with acrolein in aqueous solution can be performed according to the conditions herein disclosed.Examples

[144] Preparation of a microbially active copolymer representative of the invention

[145] Microbially active copolymers comprising an acrolein derived segment and a polyalkylene glycol oligomer obtainable by a process comprising reacting the polyalkylene glycol with acrolein in aqueous solution to form the microbially active copolymer representative of the current invention can be made according to the following references : WO2009 / 059350; WO2016 / 077879; WO2017 / 139849; and WO2021 / 151160. The polyalkylene glycol oligomer typically used in manufacture of copolymers in these applications is high purity good manufacturing practices (‘GMP’) polyethylene glycol which is suitable for human use. Such manufacture leads to a copolymer having a total ion concentration chromatogram shown in figure 11.

[146] A representative procedure for preparation of the microbially active copolymer of molecular weight using CARBOWAX® obtained from DOW chemicals containing (not suitable for human use) polyethylene glycol of average molecular weight 200, in a range of specific molecular weight of about 190 to about 210 as follows:

[147] A solution of freshly distilled or non-distilled acrolein (as noted in the table below 1) in water (20 g) was slowly added over 10 minutes to a solution of water (20 g) and polyethylene glycol (60 g; MW 200) which had been rendered pH 12 by the addition of 1M aqueous sodium hydroxide; during the 10 minutes, the yellow colour of oxidized hydroquinone quickly appeared, then disappeared. During the process the composition was continuously and vigorously stirred to provide copious contact with air. An exothermic and rapid polymerization took-place. The temperature can be maintained at 10 oC by use of a temperature-controlled heat exchanger.

[148] After another 50 minutes, the clear solution was adjusted to pH 7.5 by the addition of 1M aqueous hydrochloric acid; the product was a clear, almost colourless (very pale yellow) solution. The resultant copolymer has a maximum specific molecular weight of about 800 Daltons. The most prevalent copolymer has a specific molecular weight of about 556 Daltons. The applicant estimates the average molecular weight of the copolymer to be about 550 Daltons.

[149] Representative microbially active copolymers have the following properties.

[150] Microbially active copolymer representative of the claimed invention Batch#Microbially active copolymer 1 Microbially active copolymer 2Manufacturing   NotesModified for 1kg as described above n / aFeedstock 1 - PEG 200Polyethylene glycol molecular weight 200 (Non-technical grade)polyethylene glycol molecular weight 200 (Non-technical grade)Feedstock 2 - AcroleinDistilled in situ using atmospheric multiplate Vigreux to >99% purity prior to use.Undistilled (88% purity), as is undistilled. Contains between 0.1-0.5 wt% hydroquinone.Production Trends & ObservationsPreparation as described above.Preparation required twice the total volume of buffer(s) required to maintain the pH. Monomeric Acrolein %wt0.00044n / dHPLC Analysis Molecular weight ranges shown are the specific molecular of observed species  >2,500 Daltons0%0.752%>350 - <2,500 Daltons9.967%9.902%<350 Daltons90.029%89.346% Table 1 – Composition and analytical analysis of microbially active copolymers representative of the claimed invention

[151] The resultant copolymer compositions are difficult to examine using most analytical techniques due to the numerous and complex nature of the species of copolymers observed and as noted in Table 1. However, total ion chromatography can be used to analyse the complex mixture as follows using a “Waters Ultrahigh Performance Liquid Chromatography Mass Spectrometer (UHPLC-MS)” using a “Waters Acuity UPLC HSS T3 1.8um, 2.1 × 100mm column” attached to a “Waters Acuity QDa (single quadrupole mass spec, acquisition mode in scan mode” and “Waters Emprove 3 processing software”.

[152] The use of Carbowax leads to a copolymer having a total ion concentration chromatography shown in figure 12. Figure 12 shows structurally distinct features compared to figure 11, such as late eluting analyte peaks (after about 10 minutes for example).

[153] Without wishing to be bound by theory, the applicant speculates that use of Carbowax may lead to oligomers of a different structure than use of GMP, such as larger oligomers beyond PEG-8 for example. Again, without wishing to be bound by theory, the applicant speculates that this structural difference may lead to the beneficial properties observed for use in the amelioration of microbial growth associates with equipment.

[154] The MIC (minimum inhibitory concentration) for P.aeruginosa (PA01) was in the order of 1000s of ppm; and 100s of ppm for B.subtilus (PY79) for copolymers 1 and 2.

[155] Further aqueous biocidal compositions can be prepared by addition of a food grade gelling agent. For example, cellulose gum can be added up to about 2 wt% of the composition.

[156] Microbially active copolymers representative of the claimed invention were then subjected to the following testing.

[157] Microbial growth testing

[158] The following tests were using active undertaken in accordance with Methods as per National Association of Corrosion Engineers (NACE) TM0194-2014 (Field Monitoring of Bacterial Growth in Oil and Gas Systems).

[159] Mixed cultures were obtained from oilfield wastewaters in Australia and were cultured in the lab for a four-week period as to build key corrosion inducing populations to 10^5 to 10^7 bacteria / mL broth. Sulphur reducing bacteria (SRB) and acid producing bacteria (APB) were then measured in triplicate using most probable number (MPN), as per the TM0194-2014 standard.

[160] The APB and SRB were quantified before and after 1 hour and 24 hours of exposure with microbially active copolymer. Reductions in APB / SRB populations were then graphed as shown in the Figures in terms of log10 and percentage reductions. For example, 1 log10 reduction = 90% reduction; and 5 log10 reduction = 99.999% reduction.

[161] Planktonic Mesophilic Freshwater Simulation

[162] Conducted at 30 oC in 10,000 total dissolved salts (TDS). As shown in Figure 1, after 1 hour (upper panel) and 24 hours (lower panel) of exposure in the planktonic mesophilic freshwater simulation, microbially active copolymers representative of the currently claimed invention measured a significant reduction in SRB and APB. Thus showing that microbially active copolymers representative of the claimed invention are active in freshwater planktonic (vagile) systems at a mesophilic temperature.

[163] Planktonic Thermophilic Freshwater Simulation

[164] Conducted at 60 oC in 10,000 total dissolved salts (TDS). As shown in Figure 2, after 1 hour (upper panel) and 24 hours (lower panel) exposure in the planktonic thermophilic freshwater simulation, microbially active copolymers representative of the currently claimed invention measured significant reduction in SRB and APB. Thus showing that microbially active copolymers representative of the claimed invention are active in freshwater planktonic (vagile) systems at a thermophilic temperature.

[165] Planktonic Mesophilic Brackish Simulation

[166] Conducted at 30 oC in 25,000 total dissolved salts (TDS). As shown in Figure 3, after 1 hour (upper panel) and 24 hours (lower panel) exposure in the planktonic mesophilic brackish simulation, microbially active copolymers representative of the currently claimed invention measured significant reduction in SRB and in APB. Thus showing that microbially active copolymers representative of the claimed invention are active in brackish planktonic (vagile) systems at a mesophilic temperature.

[167] Planktonic Thermophilic Brackish Simulation

[168] Conducted at 60 oC in 25,000 total dissolved salts (TDS). As shown in Figure 4, after 1 hour (upper panel) and 24 hours (lower panel) exposure in the planktonic thermophilic brackish simulation, microbially active copolymers representative of the currently claimed invention measured significant reduction in SRB and in APB. Thus showing that microbially active copolymers representative of the claimed invention are active in brackish planktonic (vagile) systems at a thermophilic temperature.

[169] Sessile Mesophilic Freshwater Simulation

[170] Conducted at 30 oC in 10,000 total dissolved salts (TDS). As shown in Figure 5, after 1 hour (upper panel) and 24 hours (lower panel) exposure in the sessile mesophilic freshwater simulation, microbially active copolymers representative of the currently claimed invention measured significant reduction in SRB and in APB. Thus showing that microbially active copolymers representative of the claimed invention are active in sessile freshwater systems at a mesophilic temperature.

[171] Sessile Thermophilic Freshwater Simulation

[172] Conducted at 60 oC in 10,000 total dissolved salts (TDS). As shown in Figure 6, after 1 hour (upper panel) and 24 hours (lower panel) exposure in the sessile thermophilic freshwater simulation, microbially active copolymers representative of the currently claimed invention measured significant reduction in SRB and in APB. Thus showing that microbially active copolymers representative of the claimed invention are active in sessile freshwater systems at a thermophilic temperature.

[173] Sessile Mesophilic Brackish Simulation

[174] Conducted at 30 oC in 25,000 total dissolved salts (TDS). As shown in Figure 7, after 1 hour (upper panel) and 24 hours (lower panel) exposure in the sessile mesophilic brackish simulation, microbially active copolymers representative of the currently claimed invention measured significant reduction in SRB and in APB. Thus showing that microbially active copolymers representative of the claimed invention are active in sessile brackish systems at a mesophilic temperature.

[175] Sessile Thermophilic Brackish Simulation

[176] Conducted at 60 oC in 25,000 total dissolved salts (TDS). As shown in Figure 8, after 1 hour (upper panel) and 24 hours (lower panel) exposure in the sessile thermophilic brackish simulation, microbially active copolymers representative of the currently claimed invention measured significant reduction in SRB and in APB. Thus showing that microbially active copolymers representative of the claimed invention are active in sessile brackish systems at a thermophilic temperature.

[177] Comparative example

[178] Microbially active copolymers representative of the currently claimed invention were tested against commercially available example 40% aqueous glutaraldehyde. The following simulations were tested: sessile mesophilic freshwater, sessile thermophilic freshwater, sessile mesophilic brackish and sessile thermophilic brackish over 1 hour at 200 ppm, both showed greater than 99.4% reduction in SRB in each simulation. The observed activity of the that microbially active copolymers representative of the claimed invention are comparable in SRB and APB activity to those currently used commercially in this study.

[179] Aquatic toxicity testing

[180] The following tests were conducted under United Nations Globally Harmonized System of Classification and Labelling of Chemicals, Chapter 14 Hazardous to the Aquatic Environment (UN GHS).

[181] Following the protocol outlined in UN GHS microbially active copolymers representative of the current invention were tested for acute and chronic toxicity. The list of bioassays is detailed in the tables below.For quality assurance purposes reference toxicant bioassays were tested simultaneously. Statistically calculated effect concentrations (EC10, EC50, NOEC and LOEC) have been reported for the samples and reference toxicants.

[182] EC10 or EC50 is the effect concentration at which 10% or 50% effect (mortality, inhibition of growth, reproduction, etc…) is observed compared to the control group of test organisms. NOEC (No Observed Effect Concentration) is the highest tested concentration for which there is no statistically significant difference (p<0.05) when compared to the control group in long-term ecotoxicity studies. LOEC (Lowest Observed Effect Concentration) is the lowest concentration where an effect has been observed in chronic ecotoxicity studies. LOEC can be used to derive NOEC if the effect percentage of the LOEC is known. PARAMETER CONTROL 0.8 MG / L 1.6 MG / L 3.1 MG / L 6.3 MG / L 12.5 MG / L pH [1]8.21 8.2 8.2 8.2 8.2 8.2 Salinity (‰) [1]32.1 32.1 32.1 32.1 32.1 32.1 DO (%) [1]100 100 100 100 100 100

[183] Table 2– Physicochemical conditions used for aqua toxicity testingof a microbially active copolymer BIOASSAY – ACUTEPROTOCOL REFERENCE TEST SPECIES TEMP. 72-hour Microalgal Growth Bioassay WIECX-06 Stauber et al. 1994 I. galbana 22°C 48-hour Copepod Survival WIECX-26B USEPA 2002 G. imparipes 22°C 96-hour Juvenile Fish Survival WIECX-16B USEPA 2002 S.lalandi 22°C  BIOASSAY – CHRONIC PROTOCOL REFERENCE TEST SPECIES TEMP. 72-hour Microalgal Growth Bioassay WIECX-06 Stauber et al. 1994 I. galbana 22°C 5-7 day Copepod Larval Development Bioassay [1]WIECX-26 ISO 16778 G. imparipes 22°C 7-day Fish Larval Development BioassayWIECX-16 USEPA 1004.0 S.lalandi 22°C  

[184] Table 3 – bioassays used in the aqua toxicity testing of a microbially active copolymerBIOASSAY EC10 (%) EC50 (%) NOEC (%)  LOEC (%) ACUTE       72-hour Microalgal Growth 12.5  17.8 7.8  1.6 48-hour Copepod Survival [1]6.3  7.63 6.3  12.5 96-hour Juvenile Fish Survival [1]50.4  61.0 25  50 CHRONIC       72-hour Microalgal Growth 12.5  17.8 7.8  1.6 5-7 day Copepod Larval Development [1]4.2  7.01 3.1  6.3 7-day Fish Larval Development 17.5  53.1 3.1  6.3

[185] Table 4 – results of aquatic toxicity testing of a microbially active copolymer

[186] Glutaraldehyde has been described as moderately toxic aquatic organisms and as having an EC50 for 72-hour exposure to green algal growth as 1.2 mg / L and NOEC of 0.5 mg / L (for example Glutaraldehyde SDS from The Dow Chemical Company). From table 3 microbially active copolymers representative of the current invention show a nearly 15-fold improvement of up to 17.6 mg / L. The NOEC of glutaraldehyde has been described as 0.05 mg / L whereas microbially active copolymers representative of this invention have a NOEC of 7.8 mg / L requiring a factor of about 150 less to reach noticeable toxicity.

[187] Magnacide H (95% acrolein) has been described as very toxic to aquatic organisms. From table 3, microbially active copolymers representative of this invention have an EC50 for fishlings measured at 61 ppm (61,000 ppb). When compared to acrolein (1,128,363 ppb) microbially active copolymers representative of this invention requires are a factor of about 45,000 less toxic than Magnacide H for fishlings.

[188] The results in table 3 shows that microbially active copolymers representative of this invention of the present invention are well tolerated in aquatic environments as compared to commercially available current biocides based on commodity chemicals. In addition, these toxicity test results show that microbially active copolymers representative of this invention are suitable for discharge into ecosystems without significant dilution of the effective amount and / or detoxification.

[189] Physicochemical Testing

[190] Thermal stability studies

[191] Thermal stability was assessed by subjecting microbially active copolymer representative of the current invention to 65 oC for 48 hour. Any degradation or change in chemical structure was recorded using FTIR (Fourier-Transform Infrared Spectroscopy). Observations were also recorded.

[192] The following criteria were used to assess stability: visual examination for changes in colour, layer formation, and viscosity; no formation of gunk or solid; and, no change in chemical composition as measured by FTIR.

[193] Aside from some small observed changes in the chemical appearance from light-yellow to yellow colour no other changes nor development of gunk or solid were observed. As shown in Figure 9 the location and relative intensity of peaks in the FTIR spectra between the as-received and thermally-aged samples showed no significant changes. Thus microbially active copolymers were deemed to be thermally stable according to this criteria.

[194] Emulsion and Solubility Tendency

[195] The emulsion tendencies of microbially active copolymers representative of the current invention were assessed in standardized tests at ambient temperature. Hydrocarbon (Test 1, table 4) or saline simulation waters (Test 2, table 4) were placed in graduated measuring cylinders. Microbially active copolymers representative of the current invention were injected at 1,000 ppm, sealed and subjected to 100 energetic shakes before allowing for phase separation.

[196] Visual assessment was conducted over a period of one hour. The following criteria was used to assess emulsion and solubility tendency: there shall be no visible emulsion clearly visible after vigorous mixing of the chemical being tested; and if emulsion is formed, it should be dispersed (total separation) within a maximum time of 5 minutes (green). If an emulsion is present this should be dispersed within 10 minutes (yellow). Additionally, there shall be no deposit formed and no formation of separate liquid phase.SampleConcentration (ppm)Deposit formation (Y / N)Emulsion formed (Y / NTime taken for emulsion to separate Result 1 min2 minBlank control 1n / aNY0n / a<30 sPassBlank control 2n / aNY0n / a<30 sPassCopolymer 11000NY2075 sPassCopolymer 1 1000NY30101 sPass 

[197] Table 4 – observations of copolymers representative of the claimed invention recorded during this assessment

[198] After 2 minutes, microbially active copolymers representative of the current invention had no visible emulsion nor deposit for both of the washwater simulations (Test 1 Hydrocarbon 50% LAWS (Low Aromatic White Spirit)) and Test 2 (Saline) – 0.1% NaCl solution). Thus microbially active copolymers representative of the current invention show good emulsion and solubility tendency.

[199] Foaming Tendency

[200] Foaming tendency tests were conducted in 1,000mL, graduated glass measuring cylinders using synthetic brine (condensed water, 1% NaCl – Test 1, table 5) and hydrocarbon (50% LAWS* – Test 2, table 5), and by sparging the chemically 5 treated  fluids with nitrogen gas. The foaming dispersion / dissipation characteristics and relaxation period were assessed over a period of 10 minutes. Microbially active copolymers representative of the current invention were added to the hydrocarbon phase at a concentration of 1,000ppm, based on total fluids.

[201] To pass the foaming tendency test, any foaming at 50% LAWS shall collapse within 30 seconds (green) to 5 minutes (yellow).SampleConcentration (ppm)Observation after 10 minutes Foaming height (ml)Result 0 s30 sBlank control 1n / aClear, total separation 0n / aPassBlank control 2n / a Clear, total separation0n / aPassCopolymer 11000Clear, total separation1000PassCopolymer 1 1000Clear, total separation1200Pass 

[202] Table 5 - observations recorded during this assessment

[203] Microbially active copolymers representative of the current invention achieved zero foam in both the brine (1% NaCl) & hydrocarbon (50% LAWS) simulations after 30 seconds and were found to have good (little) foaming tendency.

[204] Sticky Deposit (Precipitation) Testing

[205] Tests were conducted to ascertain microbially active copolymer representative of the current invention resistance to fluid / volume loss, whilst being sparged with CO2 at 80 oC for 1hr.

[206] Volume observations (by change in mL or by percentage shown in table 6) were made after 10 minutes, 30 minutes and 60 minutes. Any loss in fluid volume or formation of sticky deposits was noted. In order to pass this test, there shall be no formation of insoluble precipitates and no change in chemical physical and / or visual appearance.Chemical DoseInitial volume10 minutes20 minutes60 minutesResults mL%mL%mL%Copolymer 1 neat10095590108020Pass – no change beside volume  

[207] Table 6 - observations recorded during this assessment

[208] There was no formation of insoluble precipitates and no change in chemical physical and / or visual appearance so microbially active copolymers representative of the current invention were found to have good (little) precipitation properties according to this test.

[209] Corrosivity Testing - Linear Polarization Resistance

[210] Test brine (1% NaCl) was prepared using analar grade chemicals and deionized water inside a glass aspirator. The synthetic hydrocarbon was added to a separate glass aspirator and both fluids were sparged with CP grade CO2 gas until the oxygen concentration was less than 10 ppb. The electrochemical probes (e.g. API 5L X65) were polished to P500, rinsed and dried and placed inside standard 1,000ml glass cells. The cells were fitted with a thermocouple, gas in and gas out tubes. The cells were then sealed and sparged with CO2 to remove traces of oxygen.

[211] The required amount of fluids (brine first) were added to the cell under positive CO2 pressure and then heated to the test temperature using proportional temperature controllers. Throughout the test duration the solution was sparged with CP grade CO2 gas. After a pre-corrosion period of 4 hours, the chemical was added to the brine and monitored changes in the corrosion rate of the LPR probe positioned in the brine phase. The following parameters were simulated in these tests: Temperature: 65 oC; Water Chemistry: Synthetic condensed water (1% NaCl); Pre-corrosion period: 4 hours; pCO2: Saturated with CO2 at test temperature; concentrations of microbially active copolymers representative of the currently claimed invention were measured at 1,000 ppm and 10,000 ppm; and corrosion monitoring: 3 electrode LPR.

[212] The material tested was API 5L X65 pipe steel and is classified as a high resistance and low alloy steel, which presents low carbon content, less than 0.30%, good tenacity and weldability. Pipelines for petroleum industry applications are constructed according to the American Petroleum Institute (API) technical specifications.Chemical Stabilized corrosion rate (mm / yr) Baseline 1000 ppm dosage 10000 ppm dosage T1T2T1T2T1T2Copolymer 1 4.325.015.535.646.125.74

[213] Table 7 - observations recorded during this assessment

[214] As shown in Figure 10 and Table 7, duplicate tests 1 and 2 showed a stable baseline at 1,000 ppm and 10,000 ppm for microbially active copolymers representative of the claimed invention. Baseline is CO2 enriched brine (1%NaCl) connected to API 5L X65 steel pipe, kept at 65 oC for 48 hours to simulate an annual corrosion rate.

[215] The observed normalized corrosion rates rate for 1,00 ppm is 0.91 mm / year; and 10,000 ppm is 1.26 mm / year. A result of around 1 mm / year considered non-corrosive and so microbially active copolymers can be considered non-corrosive according to this test.

[216] Corrosivity Testing - Immersion as per ASTM G1

[217] This test was conducted using the ASTM G1 (American Society for Testing and Materials) Standard Practice for Preparing, Cleaning, and Evaluation Corrosion Test Specimens.

[218] Industrially relevant metal coupons (carbon steel) were immersed in a CO2 enriched brine solution for a 30 day period whereafter they were retrieved and analyzed for weight loss and inspected for pitting corrosion. microbially active copolymers representative of the current invention were included at 10,000 ppm (i.e., 1%w / v)

[219] The samples were then photographed before rinsing with water for further assessment. Any corrosion residual present were cleaned using an inhibited acid solution in accordance with ASTM G1. Coupons were then re-photographed and re-weighed.

[220] Annualized corrosion rate was calculated according to ASTM G1 and are presented in table 8 and table 9.Chemical Coupon typeCoupon IDSurface area (g)Initial mass (g)Final mass (g)Mass loss (g)Duration (hours)Density (g / cm2)Corrosion rate (mm / yr)Copolymer 1 Carbon steel TC 36133.539.484338.45671.02767207.840.48TC 36233.538.227537.24170.98587207.840.48

[221] Table 8 – mass loss data from immersion testsChemical Coupon typeCoupon IDDeepest pit recorded (µm)Copolymer 1Carbon steelTC 36139.78TC 36233.18

[222] Table 9 – microscopic analysis for deepest pitting

[223] An annual corrosion rate of 0.47 mm / year and less than 40 micron pitting measured microscopically shows that microbially active copolymers representative of the current invention pass the ASTM G1 test and can be considered non-corrosive.

[224] Thermal stability studies

[225] The purpose of this study was conducted to assess non-refrigerated temperature stability of compositions representative of the claimed invention. Hydroquinone included in the final product is at level of about <0.005% w / w.

[226] Batch B0124 was manufactured in accordance with the methods disclosed herein and the initial QC results for MIC were as follows E.coli 25922 – 130-260ppm, S.aureus ATCC 29213 – 260ppm.

[227] The vials were stored in their allocated temperature storage cabinets at which coincided their labels as 5 oC – 2-8 oC, 25 oC – 25 oC and 40 C oC – 40 oC.

[228] The experiment was initiated on the 13 November 2023, with twenty vials of B0124 dispensed. Vials were individually labelled and placed into their relevant storage chamber for the duration of the trial.

[229] Table 10: MIC Results for E.coli ATCC 25922 Start1mth3mths6mths9mths12mths5oCLBI#30NOV23LBI#20DEC23LBI#22FEB24LBI#29MAY24LBI#20AUG24LBI#27NOV24B0124130-260130-260130-26065-130130-260260 Start1mth3mths6mths9mths12mths25oC------B0124N / A260130-260130260-520260-520 Start1mth3mths6mths9mths12mths40oC------B0124N / A130-260260-520260520520 

[230] Table 11: MIC Results for S.aureus ATCC 29213 Start1mth3mths6mths9mths12mths5oCLBI#30NOV23LBI#20DEC23LBI#22FEB24LBI#29MAY24LBI#20AUG24LBI#27NOV24B0124260130-260260260260260 Start1mth3mths6mths9mths12mths25oC------B0124N / A130-260260260-520260260 Start1mth3mths6mths9mths12mths40oC------B0124N / A130260-520260260-520520

[231] The stability of these preliminary samples, as shown in tables 10 and 11, after 12months at both 5 and 25oC storage, showed limited to no impact on product efficacy.

[232] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications which fall within the spirit and scope of the present invention.  

Claims

1. A method of ameliorating microbial growth associated with equipment comprising the step of: contacting the microbial growth with an effective amount of a microbially active copolymer, wherein the microbially active copolymer comprises an acrolein derived segment and a polyalkylene glycol oligomer, and wherein the microbially active copolymer is obtainable by a process comprising reacting the polyalkylene glycol with acrolein in an aqueous solution to form the microbially active copolymer.

2. The method of claim 1, wherein the equipment is used in a gas field, an oil field, a commercial environment, marine or a naval environment.

3. The method of claim 1 or 2, wherein the method further comprises the step of at least in part discharging the microbially active copolymer from the equipment into an ecosystem following contacting the microbial growth with the effective amount of the microbially active copolymer.

4. The method of claim 3, wherein discharging the microbially active copolymer from the equipment into the ecosystem is conducted without substantially diluting the effective amount or detoxifying the microbially active copolymer.

5. The method of claim 3 or claim 4, wherein the ecosystem is an aquatic ecosystem.

6. The method of any one of claims 1 to 5, wherein the microbial growth is thermophilic or mesophilic.

7. The method of any one of claims 1 to 6, wherein the microbial growth is halophilic, halotolerant or halophobic.

8. The method of any one of claims 1 to 7, wherein microbial growth is prone to cause corrosive damage.

9. The method of any one of claims 1 to 8, wherein the microbial growth is sessile or vagile.

10. The method of any one of claims 1 to 9, wherein the microbial growth is bacterial growth.

11. The method of claim 10, wherein the bacterial growth comprises acid producing bacteria or sulphur reducing bacteria.

12. The method of any one of claims 1 to 11, wherein the microbially active copolymer is formulated as a biocidal composition.

13. The method of claim 12, wherein the biocidal composition comprises unreacted free acrolein less than about 0.0001 %wt.

14. The method of claim 12 or claim 13, wherein the biocidal composition is aqueous.

15. The method of claim 14, wherein the aqueous biocidal composition has a water content is in the range of about 20 %wt to about 60 %wt water.

16. The method of any one of claims 12 to 15, wherein the biocidal composition further comprising a gelling agent or a stabilizer.

17. The method of claim 16, wherein the gelling agent is cellulose gum present in about 2 wt% or less.

18. The method of any one of claims 1 to 17, wherein the molecular weight of the polyalkylene glycol is in the range of about 100 to about 2500 Daltons.

19. The method of claim 18, wherein the molecular weight is about 200 Daltons.

20. The method of any one of claims 1 to 19, wherein the polyalkyele glycol is polyethylene glycol.

21. The method of claim 20, wherein the specific molecular weight of polyethylene glycol is about 190 to about 210.

22. The method of any one of claims 1 to 21, wherein the molecular weight of the copolymer is about 200 Daltons to 7000 Daltons.

23. The method of claim 22, wherein the molecular weight of the copolymer is about 550 Daltons.

24. The method of any one of claims 1 to 23, wherein the process is conducted at a temperature range of about -20 oC to about 50 oC.

25. The method of any one of claims 1 to 24, wherein the pH of the aqueous solution is alkaline and no more than about pH 12.5.

26. The method of any one of claims 1 to 25, wherein the effective amount is in the range of about 5 ppm to about 2000 ppm.

27. The method of any one of claims 1 to 26, wherein contacting the microbial growth with an effective amount of a microbially active copolymer is conducted between about 60 seconds to 1 hour.

28. The method of any one of claims 1 to 27, wherein the microbially active copolymer is thermally stable.

29. The method of any one of claims 1 to 28, wherein the microbially active copolymer remains substantially non-formable having been subjected to agitation.

30. The method according to any one of claims 1 to 29, wherein the copolymer is substantially chemically inert to the equipment.

31. The method of any one of claims 1 to 30, wherein the microbially active copolymer exhibits a corrosion rate of 1.00 mm / year or less at an effective amount of 1000 ppm as measured by linear polarization resistance.

32. The method of any one of claims 1 to 31, wherein the microbially active copolymer exhibits a corrosion rate of about 1.0 mm / year or less.

33. The method of any one of claims 1 to 32, wherein the microbially active copolymer exhibits an EC50 for microalgal growth of 17.8 mg / mL over 78-hours and 61 ppm juvenile fish survival over 96-hours.

34. A kit for use in generating an aqueous biocidal composition copolymer comprising a microbially active copolymer for ameliorating microbial growth, wherein the kit comprises a polyalkylene glycol and acrolein, wherein reacting the polyalkylene glycol with acrolein in aqueous solution forms the aqueous biocidal composition comprising the microbially active copolymer.

35. Use of a microbially active copolymer to ameliorate microbial growth associated with equipment, wherein the microbial growth associated with equipment is contacted with an effective amount of the microbially active copolymer, wherein the microbially active copolymer comprises an acrolein derived segment and a polyalkylene glycol oligomer, and wherein the microbially active copolymer is obtainable by a process comprising reacting the polyalkylene glycol with the acrolein in aqueous solution to form the microbially active copolymer.

36. A method of ameliorating microbial growth associated with equipment comprising the step of: contacting the microbial growth with an effective amount of a microbially active copolymer, wherein the microbially active copolymer comprises an acrolein derived segment and a polyalkylene glycol oligomer, and wherein the microbially active copolymer is obtainable by a process comprising reacting the polyalkylene glycol with acrolein in an aqueous solution to form the microbially active copolymer. 

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