WO2022118312A1

WO2022118312A1 - Method and composition for water treatment

Info

Publication number: WO2022118312A1
Application number: PCT/IL2021/051428
Authority: WO
Inventors: Michal RODENSKY; Chen Zolkov; Jakob Oren; Nir GOLDSTEIN; Ari Ayalon
Original assignee: Bromine Compounds Ltd.
Priority date: 2020-12-02
Filing date: 2021-12-01
Publication date: 2022-06-09

Abstract

The invention provides methods and compositions for the microbial control of water, in particular for eliminating planktonic and biofilm bacteria therefrom, using a bromine-based biocide in combination with a cis-2-alkenoic acid as an auxiliary agent which has been found to enhance the action of the biocide.

Description

Method and composition for water treatment

The invention relates to microbial control of water, e . g . , eliminating planktonic and biofilm bacteria using a brominebased biocide in combination with an auxiliary agent which has been found to enhance the action of the biocide .

The use of bromine in industrial water treatment is well established and a variety of bromine-based biocides are currently available in the market . The working concentrations and frequency of supply of the biocide depend on the type of water, microbial load, organic load, the speci fic biocide under consideration, the dosing method, etc .

It has been reported in WO 2008 / 143889 and the Journal of Bacteriology 191 : 1393- 1403 ( 2009 ) that cis-2-alkenoic acid ( CAA) of formula I

5 where R = C7H15, that is cis-2-decenoic acid produced by the bacterium Pseudomonas aeruginosa _r is capable of inducing P. aeruginosa and other gram-negative and gram-positive bacteria as well as fungi to undergo a physiologically-mediated dispersion response , resulting in the dis-aggregation of surface-associated microbial populations and communities known as biofilms .

Control of biofilm constitutes an important aspect of water treatment programs . In US 2009/ 0178587 , the performance of bromine-based biocides in controlling biofilms of P. aeruginosa was investigated . It has been also proposed in US 2009/ 0178587 to increase the ef ficiency of the treatment with the aid of surfactants that act as bio-dispersants , but no experimental data was given to illustrate this approach . In co-assigned PCT/ IL2020/ 050591 it was demonstrated that cis- 2-decenoic acid (CDA) can act as an effective adjunctive to bromine-containing biocides in the treatment of biofilm and planktonic bacteria in water systems and on surfaces in contact with the water, to achieve significant enhancement in the killing of bacteria in both pure and mixed cultures typically found in industrial and natural waters, relative to treatment with the brominated biocides alone.

The present invention describes the use of cis-2-alkenoic acids (CAAs) of formula I or salts thereof, other than the aforementioned cis-2-decenoic acid, that is, where R in formula I is C3-6, 8-15 alkyl (e.g., 64-6,8-11 alkyl) or mixtures thereof (CDA can be included in a mixture alongside another CAA) , as an additive in bromine-based water treatment. Therefore, unless indicated otherwise, hereinafter the term cis-2-alkenoic acid and the abbreviation CAA do not include cis-2-decenoic acid (CDA) .

Experimental results obtained by the inventors in the framework of the present invention show that adding the CAA cis-2-heptenoic acid, cis-2-undecenoic acid, cis-2-nonenoic acid or cis-2- octenoic acid to pre-grown biofilms alongside a sulfamate- stabilized bromine-based biocide improved the efficacy of the biocide in microbial control, showing a substantial log reduction of bacterial count. This effect was particularly pronounced in the presence of cis-2-heptenoic acid, where the total count reduction improved by 6.5 log units relative to a control measurement.

The invention is therefore primarily directed to a method of microbial control in water, comprising adding to the water one or more bromine-based biocide (s) and cis-2-alkenoic acid(s) (or a salt thereof) as defined above to achieve, for example, reduction of planktonic and/or biofilm bacteria, algae and fungi on a surface in contact with the water.

CAA(s) can be easily incorporated into bromine delivery systems that are currently employed in the treatment of industrial water. For example, the bromine-based biocide (s) and CAA(s) can be delivered to an industrial water stream in contact with an infested surface using multiple feed solutions injected sequentially or simultaneously, either continuously or in batch mode to the water stream; the simultaneous injection may include the pre-mixing of the individual solutions to produce a single additive solution (i.e., the CAA(s) and biocide (s) solutions can be mixed before or just prior to addition to the water stream) . The selected feeding method also depends on whether the biocide is supplied as a single component or not, as described below.

By a non-limiting example, the method of microbial control in water as herein defined comprises sequentially adding to the water one or more bromine-based biocide (s) and at least one cis- 2-alkenoic acid (6AA) of formula I, for example where the at least one cis-2-alkenoic acid (6AA) of formula I is added to the water prior to the addition of one or more bromine-based biocide ( s ) .

To enable water treatment using a single additive feed instead of multiple additives feeds, liquid concentrates comprising suitably proportioned combinations of bromine-based biocide (s) and 6AA(s) can be prepared.

Accordingly, another aspect of the invention is a composition (e.g., a liquid concentrate) comprising one or more brominebased biocide (s) and one or more cis-2-alkenoic acid(s) of formula I, where R is 63-6,8-15 alkyl (e.g., 64-6,8-11 alkyl) in a liquid carrier comprising water, water miscible solvent or mixture thereof, and optionally one or more additive (s) such as cosolvent ( s ) , antifreeze ( s ) and stabilizer ( s ) , e.g., antioxidants. Solid compositions comprising the biocide (s) and CAA(s) , e.g., granules, flakes & tablets, are also contemplated by the present invention.

Bromine-based biocides suitable for use in the present invention are available in the marketplace in different forms, i.e., solids (such as powders and compacted forms e.g., granules and tablets) and liquids (e.g., aqueous concentrates or other flowable formulations that can be easily supplied to the aqueous system to be treated) . The bromine-based biocidal agents for use in the present invention are commonly divided into two classes:

A) non-oxidizing biocides; and

B) oxidizing biocides.

Non-oxidizing biocides may be selected from the groups of:

Al: 2-bromo-2-nitro-l , 3-propanediol (bronopol) ; the synthesis of bronopol is described, for example, in WO 2009/107133. The product is available (e.g., from ICL-IP) in a powder form or an aqueous solution and its normal dose level as active ingredient (when used alone) lies in the range from 1 to 1000 parts per million (ppm) , e.g., from 1 to 300 ppm.

A2 : 2 , 2-dibromo-3-nitrilopropionamide (DBNPA) ; the synthesis of DBNPA is described, for example, in US 4,328,171. Aqueous concentrates and compacted forms of DBNPA are described in US 5, 627,135 and US 7,524,884, respectively. DBNPA is commercially available (e.g., from ICL-IP) . When used alone, dose rates as active ingredient are in the range from 1 to 1000 ppm (e.g., 1- 200 ppm) . A3: other examples of suitable non-oxidizing bromine-based biocides include 2-Bromo-4-hydroxyacetophenone (BHAP) , bis- bromo acetyl butene (BBAB) , p-bromo-p-nitro-styrene (BNS) , 2,2- dibromomalonamide and 1 , 2-Dibromo-2 , 4- dicyanobutane (DBDCB) , the preparation thereof is known in the art.

Oxidizing bromine-based biocides are compounds which release active bromine species in water (e.g., hypobromous acid/hypobromite) , either by dissolution/ dissociation or through bromide oxidation that converts the Bn to elemental bromine/Br⁺ (the oxidation is usually achieved with the aid of a chemical oxidant; however, supply of electrolytically-generated bromine to the water system to be treated is also included herein in conjunction with CAA(s) ) . The dosage of the oxidative biocides described herein is usually expressed as total CI2 that can be determined by iodometric titration using a titroprocessor : Titrino 848 plus or by DPD (Diethyl-p-PhenyleneDiamine) reagent method using a SQ-300 spectrophotometer: Merck SQ-300. Oxidizing bromine-based biocides may be selected from the groups of:

Bl: N-brominated amides and imides, such as 1 , 3-dihalo-5, 5- dialkylhydantoins , wherein at least one of the halogen atoms is bromine (the alkyl groups may be the same or different) ; commercially important biocides that belong to this class are 1- bromo-3-chloro-5, 5-dimethylhydantoin (BCDMH) , l-chloro-3-bromo-

5.5-dimethylhydantoin, 1, 3-dibromo-5, 5-dimethylhydantoin (DBDMH) and also "mixed" alkyl compounds containing two different alkyl groups at position 5 of the ring, such as 1- bromo-3-chloro-methylethylhydantoin (BCMEH) , l-chloro-3-bromo- methylethylhydantoin or mixtures thereof. Methods of synthesizing 1 , 3-dihalo-5, 5-dimethylhydantoins can be found, for example, in US 4,745,189. The acceptable dose rate of 1,3-dihalo-

5.5-dialkylhydantoins is 1 to 50 ppm as total CI2. B2 : inorganic bromide sources, namely bromide salts (e.g., alkali metal salts, ammonium bromide) and hydrobromic acid, which release bromine species in water upon oxidation (e.g., by chemical oxidation using, for example, hypochlorite, chlorine gas, hydrogen peroxide or ozone; and by electrochemical oxidation, namely, anodically-generated bromine) . Commercially important products include activated sodium bromide (consisting of an aqueous solution of sodium bromide and sodium hypochlorite prepared on-site and delivered immediately to the water system to be treated) ; activated ammonium bromide (the biocide is prepared on-site by reacting ammonium bromide with an oxidizer) ; solution of HBr and urea which reacts with e.g., sodium hypochlorite on-site (e.g., Bactebrom® solution, composed of HBr and urea, from ICL-IP; the resulting active form is sometimes named herein bromourea) ; and dry mixtures of bromide/chlorine compound that are fed, for example, in a tablet form directly into the water system to be treated to react in-situ and produce the active bromine species. The abovementioned bromide sources such as sodium bromide, hydrobromic acid, ammonium bromide and the solution of HBr (or NaBr) and urea may be oxidized on-site chemically (e.g., with hypochlorite, chlorine gas, hydrogen peroxide or ozone) or electrochemically.

B3 : Other examples of oxidizing bromine-based biocides include sulfamate-stabilized bromine-based biocides for example as described in WO 99/06320 (stabilized aqueous alkali/alkaline earth metal hypobromite solution (e.g., NaBr as bromide source) ) , or WO 03/093171, available from ICL-IP as Bromosol®, and bromine chloride and stabilized forms thereof (see US 6,068,861) available in the market as aqueous concentrates. One major example of a sulfamate-stabilized bromine-based biocide is an aqueous solution of alkali hypobromite (e.g., NaOBr) stabilized by sulfamic acid or a salt thereof. First, the alkali hypobromite is prepared, either by the reaction of a water- soluble bromide source, such as NaBr, with alkali hypochlorite, such as NaOCl; or by addition of elemental bromine to aqueous alkali hydroxide solution (~30 wt . % NaOH solution) . Next, sulfamic acid, usually in the form of the in-situ prepared sodium sulfamate salt, is added to the hypobromite solution. The pH of this sulfamate-stabilized hypobromite solution is strongly alkaline (>10, e.g., >11 or >12) , with Br2 levels in the range from e.g., 5 to 20%, 10 to 20%, 15 to 18% and 16 to 18% Br2. Bromosol®, a commercial product tested in the studies reported below, is the reaction product of Br2 and NaOH as described above, to which sodium sulfamate was added, with pH and bromine level indicated above. The experimental results provided herein indicate that sulfamate-stabilized hypobromite solution as described above, in combination with cis-2-heptenoic acid, cis- 2-undecenoic acid, cis-2-nonenoic acid or cis-2-octenoic acid, especially cis-2-heptenoic acid, is effective against biofilm and the use of these combinations form preferred embodiments of the invention. Another major example of sulfamate-stabilized bromine-based biocides is an aqueous solution of bromine chloride, added to sodium sulfamate, usually concurrently with sodium hydroxide.

Turning now to cis-2-alkenoic acid(s) , it can be used as pure oil dissolved in a suitable solvent, such as ethanol. The cis- 2-alkenoic acids R-CH=CH-COOH of formula I for use by the invention are preferably linear. That is, R is usually a straight alkyl chain CH3- (CH2)_n- (3<n, e.g., 3<n<5 and 7<n<14, e.g., 7<n<10) . The invention contemplates the use of pure cis-2- alkenoic acid(s) as well as their use in a crude form, e.g., 80- 95% (by gas chromatography (GC) , area %) . The lower than 95% (<95%) pure CAA(s) is named herein "low purity CAA(s) grade". The term "pure CAA(s)" refers to CAA(s) characterized in having a purity level of more than 95%, e.g., equal to or greater than 97% as detected by GC . Cis-2-alkenoic acid(s) for use by the present invention are commercially available, or can be prepared for example by a two-step synthesis consisting of brominating the corresponding 2-alkanone to give crude 1 , 3-dibromo-2-alkanone as a main product alongside other isomers, followed by rearrangement of the 1 , 3-dibromo-2-alkanone to the unsaturated acid, depicted by the scheme below:

Br Favorskii

Bromination L rearrangement

Br

O O

(where R' is alkyl, e.g., C2H5, C3H7, C4H9, C₆HI₃, C7H15, C₈HI₇ and C9H19, etc.) The abovementioned two-step synthesis was first described by Rappe et al. (Acta Chemica Scandinavica (1965) , Vol. 19 p. 383-389) . The rearrangement took place in an alkaline environment, using alkali carbonate or alkali bicarbonates as a base. A similar approach was reported by the same research group in Organic Syntheses (1973) , Vol. 53, p.123-127. In US 8,748,486, the same synthetic pathway is described, but with alkali hydroxide in place of carbonates/bicarbonates .

A beneficial synthetic pathway arriving at cis-2-alkenoic acid is further illustrated by the scheme shown below, where 1 , 3-dibromo-2-alkanone is rearranged in an alkaline solution of potassium carbonate to the corresponding acid (in the form of potassium salt) , in the presence of a catalytically effective amount of the potassium salt of the cis-2-alkenoic acid. That is, 1 , 3-dibromo-2-alkanone is added to a reaction vessel which was previously charged with aqueous K2CO3 (10% to 30% w/w) and ~l-5 molar % of the potassium salt of the cis-2-alkenoic acid (obtained, for example, from an earlier rearrangement reaction; indicated "AP-RM" below the arrow in the scheme) : Br in water at 50°C

+ 3K2CO3 + H2O

2-3 hours

in the presence of AP-RM

Cis-2-alkenoic acid, in the form o f the free acid, is obtained by acidi fication ( e . g . , with concentrated hydrochloric acid) of the aqueous phase of the reaction mixture , i . e . , after phase separation .

A few illustrative 1 , 3-dibromo-2-alkanones which can undergo the rearrangement reaction depicted above , to give crude cis- 2-alkenoic acids for use in the present invention are tabulated in Table 1 below, along with the characteristic purity levels of the crude acid ( the list below is illustrative ; homologues having a longer carbon chain can also be used in bromine-based water treatments of the invention) :

Table 1

The 1 , 3-dibromo-2-alkanones are accessible by brominating the corresponding 2-alkenone in concentrated hydrobromic acid by slow addition of bromine , as illustrated below for the preparation of 1 , 3-dibromo-2-nonanone (1,3-DBN) and 1,3-dibromo-

2-undecanone (1,3-DBUD).

Accordingly, another aspect of the invention is a method of microbial control in water, which comprises combatting planktonic bacteria and/or biofilm bacteria on a surface in contact with the water and/or inhibiting biofilm formation on a surface prone to such formation, by adding to the water an effective microbiocidal amount of the bromine-based biocide (s) and an enhancement-inducing amount of the cis-2-alkenoic acid(s) to achieve biofilm reduction which is at least 1 log unit, e.g., at least 2, 3 or more log units higher than the reduction achieved with the same dosage of the biocide acting alone, for example, down to <10⁵ CFU/cm², e.g., <10³ CFU/cm² and preferably <10² CFU/cm² or even substantial biofilm eradication, i.e. <10² CFU/cm².

For example, the methods as herein defined are for controlling

Pseudomonas aeruginosa.

The effective microbiocidal amount of the bromine-based biocide(s) is from 0.1 to 1000 ppm, e.g., 0.1 to 300 ppm as active biocide, for example, 0.2 to 100 ppm; 0.5 to 100 ppm, and the enhancement-inducing amount of CAA(s) is from 1 nM to 30 mM. It should be borne in mind that dosage levels may vary broadly depending on factors such as the identity of biocide and intended use. But in general, effective dosing ratios biocide : CAA(s) as w/w in the water stream may vary in the range from 20:1 to 5000:1 preferably from 100:1 to 3000:1. The enhancement-inducing amount of CAA(s) can be determined by trial and error in the site of use to achieve targeted biofilm reduction. For example, an enhancement-inducing amount of CAA(s) could be from 0.001 to 15 ppm, e.g., from 0.005 to 10 ppm, for example, from 0.01 to 10 ppm, e.g., across the range of 0.005 to 0.5 ppm.

The present invention is particularly directed to provide microbial control over Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus mycoides , Candida albicans , Aspergillus niger, and combinations of microorganisms growing in mixed-species communities derived from an industrial or an environmental water source .

Figure 1 schematically illustrates one convenient method to feed a bromine-based biocide and CAA into an industrial water system. The water stream that comes in contact with a biofilm surface or a surface prone to biofilm formation is indicated by numeral (1) . The term "industrial water" is used to indicate any aquatic industrial water treatable by a bromine-based biocide according to the methods of the present invention, for example, recirculating and once-through cooling systems, cooling towers, pulp and paper mill systems, membranes, oil & gas applications, including biodiesel and diesel, floating production storage and offloading (FPSO) systems, sulphate reduction units (SRU) , steel mills, sugar & ethanol production, dairy production, swimming pools and spas, water distribution systems, irrigation systems, air washers, evaporative condensers, scrubbing systems, brewery pasteurizers, decorative fountains and oil recovery injection water .

It is seen that in the specific design illustrated in Figure 1, the biocide and CAA are held separately in tanks (2) and (3) , respectively, with their supply to the industrial water stream being accomplished by using two dosing pumps (2p and 3p) . The design enables either sequential or simultaneous application of the two active components. Biocides which fit well into the method shown in Figure 1 are biocides which are applied as a single pumpable formulation, for example, non-oxidizing biocides available in the marketplace as storage stable liquid formulations, e.g., concentrated bronopol and DBNPA solutions (e.g., 5 to 50 wt% concentrates) , and stabilized solutions of bromine or hypobromite (e.g., sulfamate- stabilized bromine-based biocide) .

The design shown in Figure 1 can be modified to enable the use of hypobromite-based biocidal solutions prepared on-site by oxidizing the bromide source just prior to use (these solutions must be applied immediately due to the instability of the hypobromite) , by installing a third feed system into the process (i.e., one dosing pump is dedicated for supplying the CAA and two dosing pumps are used for the individual components of the biocide, i.e., the bromide source and the oxidant) .

Incorporation of CAA(s) into water treatments where the bromine based-biocide is applied in solid forms such as granules or tablets (fed to the inflow water line through erosion feeders) could be achieved by injecting the CAA(s) solution with the aid of a dosing pump to the water line or to a subsidiary water stream diverted from the main stream into the feeder to dissolve the added solids.

The biocide (s) and CAA(s) solutions are dosed with metering pumps (2p and 3p, respectively) controlled by timers set up according to the treatment program. The biocide (s) and CAA(s) feed solutions may be injected directly to the water stream (1) but premixing of the two individual solutions in a mixing chamber (not shown) and delivery of the combined solution to the water stream is also possible to enable a treatment program based on simultaneous application of the two components of the treatment. To better control the treatment, monitoring and upstream mixing (4) devices are included, namely, halogen monitoring, oxidation reduction potential (ORB) , pH sensors and online static mixers.

Regardless of the exact design, the separately supplied CAA(s) can be applied neat or dissolved in a water miscible solvent or mixture of solvents such as aliphatic alcohols up to 4 carbons, tert-butyl methyl ether (MTBE) , tetrahydrofuran (THE) , dimethyl sulfoxide (DMSO) , glycols and polyethylene glycols, acetonitrile, optionally in the presence of surfactants and stabilizers.

Accordingly, the invention provides a method wherein the bromine-based biocide (s) and CAA(s) are supplied to an industrial water stream in contact with an infested surface using multiple feed solutions, whereby the biocide (s) and CAA(s) are added sequentially or simultaneously to the water.

In operation, sequential treatment with cis-2-alkenoic acid(s) can be performed by injecting the cis-2-alkenoic acid(s) from 20 minutes to 24 hours or more, prior to the biocide application. Cis-2-alkenoic acid(s) may also be added following the biocide application to enhance the activity of the residual biocide in a water sample any time over the period of time that the active biocide is present in a system.

The method of the invention does not necessarily require multiple feeds as shown in Figure 1. For example, CAA(s) and either the precursor of oxidative biocide (inorganic bromide sources) , sulfamate-stabilized bromine-based biocides or non-oxidizing biocides can be co-formulated in a liquid concentrate, e.g., with the aid of suitable stabilizers (antioxidant such as butylated hydroxytoluene (BHT) ) . Accordingly, the invention also provides a method wherein the bromine-based biocide (s) and CAA(s) are supplied to an industrial water stream in contact with an infested surface using a single feed solution, whereby the biocide and CAA are added simultaneously to the water. Thus, the invention relates to a composition comprising one or more bromine-based biocide (s) and cis-2-alkenoic acid(s) or a salt(s) thereof (e.g., for use in the method) .

For example, a nonoxidizing bromine-based biocide (s) and CAA(s) are formulated in a liquid concentrate, which is supplied to the industrial water stream using a single feed solution.

The liquid concentrates of the present invention comprise: a suitably proportioned mixture of (one or more) nonoxidizing bromine-based biocide (s) and cis-2-alkenoic acid(s) (or a salt(s) thereof) , e.g., at a weight ratio from 1000:1 to 20:1, preferably from 500:1 to 20:1, e.g., from 250:1 to 20:1, such that on dilution in an industrial water stream the two active components are applied at an effective ratio; for example, in the liquid concentrate, the concentration of the biocide is from 2 to 50%, preferably from 10 to 50% and the concentration of cis-2-alkenoic acid(s) is from 0.05 to 2%, preferably from 0.1 to 1.0% (by weight based on the total weight of the liquid concentrate) ; and a carrier comprising water, water miscible solvent or a mixture thereof (i.e., water alone, organic solvent alone or aqueous/organic solvent system) ; and optionally one or more of the following additive components: cosolvents (e.g., glycols in which the nonoxidizing bromine-based biocide exhibits high stability and solubility, e.g., ethylene glycol, propylene glycol, or dipropylene glycol monomethylether) , antifreezes and stabilizers (e.g., an antioxidant, such as butylated hydroxytoluene) . The concentrates are readily prepared by combining cis-2- alkenoic acid(s) , the nonoxidizing bromine-based biocide in a solid form, the glycol, water and the stabilizer under stirring at room temperature to obtain a clear solution.

Figure Legends

Figure 1 is a schematic illustration of an exemplary feeding of a bromine-based biocide and CAA into an industrial water system. Figure 2 is a bar diagram showing the average bacterial load per area (in colony forming units (CFU) per cm², in logarithmic scale) in a biofilm based on Pseudomonas aeruginosa that was treated with phosphate buffer (control) , cis-2-heptenoic acid, Bromosol® and a combination of Bromosol® and cis-2-heptenoic acid at the indicated concentrations.

Figure 3 is a bar diagram showing the average bacterial load per area (in colony forming units (CFU) per cm², in logarithmic scale) in a biofilm based on Pseudomonas aeruginosa that was treated with phosphate buffer (control) , cis-2-undecenoic acid, Bromosol® and a combination of Bromosol® and cis-2-undecenoic acid at the indicated concentrations .

Figure 4 is a bar diagram showing the average bacterial load per area (in colony forming units (CFU) per cm², in logarithmic scale) in a biofilm based on Pseudomonas aeruginosa that was treated with phosphate buffer (control) , cis-2-nonenoic acid, Bromosol® and a combination of Bromosol® and cis-2-noneoic acid at the indicated concentrations.

Figure 5 is a bar diagram showing the average bacterial load per area (in colony forming units (CFU) per cm², in logarithmic scale) in a biofilm based on Pseudomonas aeruginosa that was treated with phosphate buffer (control) , cis-2-octenoic acid, Bromosol® and a combination of Bromosol® and cis-2-octenoic acid at the indicated concentrations . Examples

Bromine-containing biocides suitable for use in the invention are tabulated in Table 2 below:

Table 2

*As active biocide

GC for determining purity of crude CAA Gas-chromatograph HP 7890A was used.

Method: Initial temp. 50°C, held 2 min, then raised to 280°C at 10°C/min and held for 5 min, then raised to 300°C at 10°C/min and held for 2 min.

Injector: 250°C

Detector: 300°C

Split ratio: 1:40

Concentration of the product sample: ~20 mg/ml DCM

Injection amounts: 1 .1 sample

Column: Agilent J&W Columns, HP-5, 30 m x 0.32 mm x 0.25|i Part no. 19091J-413, Ser. No. USF302346H Preparation 1

Biocide preparation by activation of HBr/urea solution

Stock solution 1 - 8.94 g of Bactebrom® from ICL-IP (HBr : urea solution) diluted with 241.06 g of distilled water.

Stock solution 2 - NaOCl ~1% prepared by 23.58g of NaOCl 10.6% w/w diluted with 226.42 g of distilled-water .

Stock solution 2 (250.00 g of NaOCl 1.0%) was added gradually while stirring to the above diluted Bactebrom® solution (stock solution 1) , to get the active biocide (orange solution) - total weight 500.00g. Expected biocide concentration as determined by iodometric titration using Titroprocessor: Titrino 848 plus. : ~ 0.5% as CI2 (~5000 ppm as CI2) . Desired biocide concentrations can be obtained by dilution with distilled water.

Preparation 2

Biocide preparation by activation of ammonium bromide

975 pl 10.25 Wt% aq. NaOCl was diluted with distilled water to 100 ml in a volumetric flask. CI2 concentration was ~1000ppm as CI2 as determined by iodometric titration using Titroprocessor : Titrino 848 plus.

213 mg NJhBr was diluted with distilled water to 100 ml in a volumetric flask.

Mix equal volumes of 5 ml as follows: add the NaOCl solution in one stroke to a mixed solution (using a magnetic stirrer) of the NJhBr solution at ambient temperature.

The concentration of the product (activated AmBr) was based on the concentration of the Na- Hypochlorite (~1000 ppm as CI2) .

Equal volumes of the reactants were mixed to obtain the concentration of the active chlorine in the mixture as 50% of the concentration of the reactant NaOCl, ~500 ppm as CI2. Desired biocide concentrations can be obtained by dilution with distilled water.

Preparation 3

Preparation of cis-2-nonenoic acid

Step 1 (brominating 2-nonanone) :

Into a mixture of 2-nonanone (from Sigma-Aldrich; 182 g, 1.28 mol) and aq. 48% HBr (300 g) , stirred and cooled to ~10°C, was added bromine (410 g, 2.56 mol) , dropwise over 3 hours. The reaction started immediately with the start of the addition of the bromine and no accumulation of bromine was observed. The reaction was exothermic and accompanied by the emission of HBr gas, just before the end of the addition of the bromine, which was absorbed in a scrubber.

Most of the reaction took place during the addition of the bromine and cooking at room temperature (~20°C) for 2.0 hours. After leaving overnight (~17 h) at room temperature, with stirring, the composition of the reaction mixture stabilized. Partial conversion of the 3 , 3-dibromo-2-nonanone (3,3-DBN) to the desired product, 1 , 3-dibromo-2-nonanone (1,3-DBN) , took place. To the reaction mixture was added water (160 g) at RT, with stirring for 30 min, and the phases were separated.

An aqueous phase (624 g) was obtained containing ~50% HBr (d = 1.50 g/ml) and crude DBN (382 g, d = 1.47 g/ml) . The concentration of 1,3-DBN in the crude product was 70.6% (GC, area % ) .

Step 2 (rearranging 1 , 3-dibromo-2-nonanone (1,3-DBN) ) :

An aqueous solution of K2CO3, in a concentration of 25% w/w, was prepared in a IL stirred reactor by the batchwise addition of K2CO3 (200 g) to water (600 g) . The reaction was exothermic. The clear solution obtained was heated to 46°C and crude DBN from Step 1 (191 g) was added to it dropwise over 45 min. The progress of the reaction was monitored by the change in the pH and the TR.

Based on the pH (unchanged at ~13) and on GC, it was seen that no reaction had taken place during the addition of the crude DBN. Immediately after the addition of the crude DBN, the pH started to go down and the TR started to go up.

The end of the reaction was determined by the pH (drop in the pH from 13.3 to 9.1) and by GC analysis of the reaction mixture (disappearance of 1,3-DBN to <1% , area %) . The phases were separated. The organic phase (42.6g) was organic waste.

In order to reduce the amount of impurities to a minimum, the aqueous phase (948 g) was washed three times with dichloromethane (DCM, 3 x 250 g) .

After the washing stage, an aqueous phase was obtained containing cis-2-nonenoic acid potassium salt (CNA-K) , organic by-products, KBr and KHCO3. In order to obtain the crude cis- 2-nonenoic acid (CNA) , the aqueous phase was acidified by the dropwise addition of aq. 32% HC1 (227 g) over 1 h. During the acidification, CO2 was emitted.

After stopping the stirring, an aqueous phase (978 g) was obtained containing salts: KC1 and KBr (heavy phase, d = 1.19 g/ml) and wet crude CNA (light phase, 51 g, d = 1.02 g/ml) which was analysed by GC and ¹H-NMR. The purity of the obtained CNA was 92.0% (by GC, area %) . Evaporation of the DCM and lights from the wet CNA under vacuum (at TB = 50°C) gave crude CNA (46.6 g) .

Preparation 4

Preparation of cis -2 -undecenoic acid

Step 1 (brominating 2-undecanone ) :

Into a mixture of 2-undecanone (218 g, 1.28 mol) and aq. 48% HBr (300 g) , stirred and cooled to ~10°C, was added bromine (410 g, 2.56 mol) , dropwise over 3 hours. The reaction started immediately with the start of the addition of the bromine and no accumulation of bromine was observed. The reaction was exothermic and accompanied by the emission of HBr gas, just before the end of the addition of the bromine, which was absorbed in a scrubber.

Most of the reaction took place during the addition of the bromine and cooking at room temperature (~20°C) for 3.5 hours. After standing overnight (~16.5 h) at room temperature, without stirring, the composition of the reaction mixture stabilized. Partial conversion of the 3 , 3-dibromo-2-undecanone (3,3-DBUD) to the desired product, 1 , 3-dibromo-2-undecanone (1,3-DBUD) , took place. To the reaction mixture was added water (160 g) at RT, with stirring for 30 min, and the phases were separated.

An aqueous phase (627 g) was obtained containing ~50% HBr (d = 1.50 g/ml) and crude DBUD (415 g, d = 1.39 g/ml) . The concentration of 1,3-DBUD in the crude product was 69.1% (GC, area%) .

Step 2 (rearranging 1 , 3-dibromo-2-undecanone (1,3-DBUD) ) :

DBUD is rearranged to cis-2-undecenoic acid (CUDA) by the following reaction:

Preparation 5

Preparation of cis-2-heptenoic acid

Step 1 :

Into a mixture of 2-heptanone (from Sigma-Aldrich; 146 g, 1.28 mol) and aq. 48% HBr (300 g) , stirred and cooled to ~10°C, was added bromine (410 g, 2.56 mol) , dropwise over 3 hours. The reaction started immediately with the start of the addition of the bromine and no accumulation of bromine was observed. The reaction was exothermic and accompanied by the emission of HBr gas, just before the end of the addition of the bromine, which was absorbed in a scrubber.

Most of the reaction took place during the addition of the bromine and cooking at room temperature (~20°C) for 4.5 hours. After standing overnight (~17 h) at room temperature, with stirring, the composition of the reaction mixture stabilized. Partial conversion of the 3 , 3-dibromo-2-heptanone (3,3-DBH) to the desired product, 1 , 3-dibromo-2-heptanone (1,3-DBH) , took place. To the reaction mixture was added water (160 g) at RT, with stirring for 30 min, and the phases were separated.

An aqueous phase (631 g) was obtained containing ~50% HBr (d = 1.52 g/ml) and crude DBH (351 g, d = 1.60 g/ml) . The concentration of 1,3-DBH in the crude product was 72.6% (GC, area%) . Step 2 :

An aqueous solution of K2CO3, in a concentration of 25% w/w, was prepared in a IL stirred reactor by the batchwise addition of K2CO3 (200 g) to water (600 g) . The reaction was exothermic. The clear solution obtained was heated to 49°C and crude DBH from Step 1 (173 g) was added to it dropwise over 1 h. The progress of the reaction was monitored by the change in the pH and the TR.

Based on the pH (unchanged at ~13) , it was seen that no reaction had taken place during the addition of the crude DBH. Immediately after the addition of the crude DBH, the pH started to go down and the TR started to go up.

The end of the reaction was determined by the pH (drop in the pH from 13.5 to 9.3) and by GC analysis of the reaction mixture (disappearance of 1,3-DBH to <1% , areal) . After completion of the reaction, cooling to RT and stopping the stirring, an organic phase appeared above the aqueous phase which contained unreacted 3-BH and 3,3-DBH, and by-products formed by a condensation reaction of crude DBH. The phases were separated. The organic phase (24 g) is organic waste.

Before starting the washings, water (50 g) was added to the reaction mixture (948g) . In order to reduce the amount of impurities to a minimum, the diluted reaction mixture (998g) was washed three times with dichloromethane (DCM, 3 x 250 g) .

After the washing stage, an aqueous phase was obtained containing cis-2-heptenoic acid potassium salt (CHA-K) , organic by-products, KBr and KHCO3. In order to obtain the crude cis-2- heptenoic acid (CHA) , the aqueous phase was acidified by the dropwise addition of aq. 32% HC1 (193 g) over 1 h. During the acidification, CO2 was emitted. After stopping the stirring, an aqueous phase (1014 g) was obtained containing salts: KC1 and KBr (heavy phase, d = 1.18 g/ml) and wet crude CHA (light phase, 45 g, d = 1.00 g/ml) which was analysed by GC and ¹H-NMR (see Figures 6A, 6B and 6C for ¹H-NMR spectra) . The purity of the CHA obtained was 95.6% (by GC, area%) .

Evaporation of the DCM and lights from the wet CHA under vacuum

(at T_B = 50°C) gave crude CHA (44 g) .

Preparation 6

Preparation of cis-2-octenoic acid

Step 1 :

Into a mixture of 2-octanone (from Sigma-Aldrich; 164 g, 1.28 mol) and aq. 48% HBr (300 g) , stirred and cooled to ~10°C, was added bromine (410 g, 2.56 mol) , dropwise over 3 h. The reaction started immediately with the start of the addition of the bromine and no accumulation of bromine was observed. The reaction was exothermic and accompanied by the emission of HBr gas, just before the end of the addition of the bromine, which was absorbed in a scrubber.

Most of the reaction took place during the addition of the bromine and cooking at room temperature (~20°C) for 2.5 hours. After leaving overnight (~15 h) at room temperature, with stirring, the composition of the reaction mixture stabilized. Partial conversion of the 3 , 3-dibromo-2-octanone (3,3-DBO) to the desired product, 1 , 3-dibromo-2-octanone (1,3-DBO) , took place. To the reaction mixture was added water (160 g) at RT, with stirring for 30 min, and the phases were separated. An aqueous phase (636 g) was obtained containing ~50% HBr (d = 1.51 g/ml) and crude DBO (358 g, d = 1.54 g/ml) . The concentration of 1,3-DBO in the crude product was 71.0% (GC, area%) .

Step 2 :

An aqueous solution of K2CO3, in a concentration of 25% w/w, was prepared in a IL stirred reactor by the batchwise addition of K2CO3 (200 g) to water (600 g) . The reaction was exothermic. The clear solution obtained was heated to 49°C and crude DBO from Step 1 (182 g) was added to it dropwise over 1 h. The progress of the reaction was monitored by the change in the pH and the

TR.

Based on the pH (unchanged at ~13) and on GC, it was seen that no reaction had taken place during the addition of the crude DBO. Immediately after the addition of the crude DBO, the pH started to go down and the TR started to go up.

The end of the reaction was determined by the pH (drop in the pH from 13.7 to 9.3) and by GC analysis of the reaction mixture (disappearance of 1,3-DBO to <1% , area%) .

Before starting the washings, water (75 g) was added to the reaction mixture (982 g) . In order to reduce the amount of impurities to a minimum, the reaction mixture was washed four times with dichloromethane (DCM, 4 x 250 g) .

After the washing stage, an aqueous phase was obtained containing cis-2-octenoic acid potassium salt (COA-K) , organic by-products, KBr and KHCO3. In order to obtain the crude cis-2- octenoic acid (COA) , the aqueous phase was acidified by the dropwise addition of aq. 32% HC1 (178 g) over 1 h. During the acidification, CO2 was emitted. After stopping the stirring, an aqueous phase (938 g) was obtained containing salts: KC1 and KBr (heavy phase, d = 1.18 g/ml) and wet crude COA (light phase, 44 g, d = 1.00 g/ml) which was analysed by GC and ¹H-NMR (see Figures 5A, 5B and 5C for ¹H-NMR spectra) . The purity of the COA obtained was 89.6% (by GC, area%) .

Evaporation of the DCM and lights from the wet COA under vacuum (at T_B = 50°C) gave crude COA (41.3 g) .

A protocol for measuring enhancement of bromine-based water treatment by addition of CAA

(3-day biofilm, simultaneous application)

The effect of bromine-containing biocide in combination with CAA on pre-grown biofilms is studied by the following experimental protocol, utilizing (A) the P. aeruginosa strain PA14 and (B) mixed bacterial species derived from environmental and industrial water.

Bacteria are cultured in EPRI medium supplemented with Hutners mineral solution and glucose (0.2%) . Microorganisms are incubated at room temperature (22°C) , under aerobic conditions with shaking. A biofilm culture system includes polystyrene 24- well plates that are treated with protein to enhance attachment and growth of biofilm bacteria according to the method described in Davies DG, Marques CN, 2009, J Bacteriol 191:1393-1403.

Following inoculation with 1 mL bacterial culture, spent medium is removed and replaced with sterile medium every 24 hours for 3 days, and a final medium exchange is performed 3 hours prior to treatment. Additionally, the medium is exchanged prior to treatment in order to remove planktonic bacteria. A treatment consists of 100 pL of 310 nM CAA and brominecontaining biocide (or bromine-containing biocide alone in the comparative treatment) , at concentrations used in commercial water treatment (e.g., from 5 to 15 ppm) , water can be used as a carrier, for a contact time determined by activity of each biocide and ranging from 1 hour to 24 hours.

Following the treatment, the medium from each well is removed by pipet and 1 mL of DE neutralization broth is added in order to stop the treatment. Bacteria from each well are then removed by scraping the biofilm formed in the well with a sterile cellscraper, and 1.0 mL of culture is transferred to chilled (4°C) 9.0 mL DE neutralizing broth and homogenized for 15 seconds at 40,000 rpm on ice. Further dilutions are performed prior to enumeration (further neutralizing biocide activity) . Recovery of bacteria is tested at different dilutions of biocide in water, in LB medium with thioglycolate and in DE neutralizing broth to ensure the active agent has been properly neutralized. Viable bacteria are enumerated via the drop plate method.

Each bromine-containing biocide is evaluated using 24-well plates, equally divided to control cultures (Ctl) inoculated with P. aeruginosa but not treated, a CAA minus test (-CAA) treated with bromine-containing biocide only, and a CAA plus test treated with bromine-containing biocide and CAA (+CAA) .

A protocol for measuring enhancement of bromine-based water treatment by addition of CAA (3-day biofilm, sequential application)

The effect of the sequential addition of CAA and brominecontaining biocide is studied using P. aeruginosa biofilm grown in the CDC biofilm reactor on borosilicate glass coupons (test method E2562 ) . The addition of the bromine-containing biocide takes place 60 min after the addition of 31nM, 310nM and 3100nM CAA. For testing the ef ficacy of the treatment against mixed biofilm, two more bacteria strains ( staphyl ococcus aureus 6538 , bacill us mycoides 6462 ) are added to the reactor .

The ef ficacy test on the coupons is performed according to the single tube method (E2871- 13 ) . This test method is used for growing a reproducible P. aeruginosa biofilm in a CDC Biofilm Reactor .

Biofilm formation :

The biofilm is established by operating the reactor in a batch mode (no flow of the nutrients ) for 4 hours . A steady state population is reached after the reactor operated for an additional 3 days with continuous flow of the nutrients . During the entire 3-day period, the bio film is exposed to continuous fluid shear from the rotation of a baf fled stir bar . At the end of the 3 days , the biofilm from the coupons is sampled as follows : a . The coupons are rinsed to remove planktonic cells . The rods are oriented in a vertical position directly over a 50 mL conical centri fuge tube which contains 30 mL sterile buf fered water . The rods are immersed with a continuous motion into the buf fered water with minimal to no splashing, then immediately removed . A new 50 mL conical tube containing 30 mL sterile buf fered water is used for each rod . b . The rods are held with one of the randomly selected coupons centered over an empty, sterile 50 mL conical tube . The set screws are loosened, allowing the coupons to drop directly to the bottom of the tube . Sequential addition of CAA and bromine-containing biocide: a. Four mL of a solution containing either phosphate buffer (untreated control) , a buffer with 310 nM CAA or a buffer with different concentrations of biocides is slowly pipetted into the tubes containing the coupons. b. Each tube is tapped to release any air bubbles trapped below the coupon. c. The tubes (containing the control, CAA or the biocide) are incubated at 20 °C, under shaking at 200 rpm for one hour contact time . d. After one hour contact time, 36 ml of a neutralizer are added to each tube. e. The combined treatment is carried out as a sequential treatment in which CAA is introduced first, and after 1 hour contact time with CAA, the coupons are transferred to another tube containing the biocide and incubated at 20 °C, under shaking of 200 rpm for another 1 hour contact time. After the one-hour contact time, 36 ml of a neutralizer are added to each tube.

Removing and Disaggregating the Biofilm: a. Each tube is vortexed using Vortex Genie-2 Model no. G560E on the highest setting for 30 s. b. The tubes are sonicated at 45 kHz for 30 s. c. The tubes are vortexed as described above, then sonicated and vortexed again. d. The samples are serially diluted in buffered water. e. Each dilution is cultured in duplicate (on R2A agar) for colony growth using the pour plating method. f. The plates are incubated at 35°C for 72 h. g. The number of colonies is counted. Example 1 Enhancing the effect of a bromine-based biocide on massive biofilm with the aid of cis-2-heptenoic acid

The ef fect of bromine-containing biocides in combination with cis-2-heptenoic acid ( also termed herein "CHA" ) on pre-grown biofilms was studied, by a sequential application of the fatty acid and the biocidal agent , as detailed below .

Experimental procedure :

Biofilms were grown using the CDC Biofilm Reactor, as detailed above , utili zing the Pseudomonas aeruginosa strain ATCC 700888 . Next , the Single Tube Method described above was used to evaluate the ef ficacy of the agents used against the Pseudomonas aeruginosa biofilm, alone or in combination, according to the sequential application protocol described above . Briefly, the single tube experiment was carried out by first adding the fatty acids solution to the tube , at a concentration of 310 nM, for one hour . After one hour , the treated coupon was moved to a second tube , containing a biocide solution in the desired concentration .

The bromine-based biocide tested in this study was Bromosol® ( stabili zed bromine ) .

Results

The results of biofilm treatment with Bromosol® in the absence or in the presence of cis-2-heptenoic acid are presented in Figure 2 , which demonstrate that addition of cis-2-heptenoic acid solution to the biofilm cultures improved the ef ficacy of Bromosol® when used at 5 mg/L ( 5 ppm) .

As shown in Figure 2 , the biocide Bromosol® ( at 5 mg/L or 5 ppm) when applied alone , reduced the total count of bacteria in the biofilm by about three log units . However, by adding cis-2- heptenoic acid at a concentration of 310 nM for 1 hour prior to the addition of Bromosol®, the total count reduction improved by additional ~3 log units with respect to the ef fect of the biocide acting alone . In other words , in the presence of the combination of the biocide and cis-2-heptenoic acid, a total count reduction of about 6 . 5 log units as compared to the control was obtained .

Example 2 Enhancing the effect of a bromine-based biocide on massive biofilm with the aid of cis-2-undecenoic acid

The inventors have next examined the ef fect of cis-2-undecenioc acid ( CUDA) in improving the ef ficacy of various biocides on biofilm removal and prevention, as detailed below .

Experimental procedure :

Biofilms were grown using the CDC Biofilm Reactor, as detailed above , utili zing the Pseudomonas aeruginosa strain ATCC 700888 . Next , the Single Tube Method detailed above was used to evaluate the ef ficacy of the agents used against the Pseudomonas aeruginosa biofilm alone or in combination, according to the sequential application protocol described above .

Speci fically, the single tube experiments were performed by first adding the fatty acids solution to the tube in the desired concentration for one hour . After one hour the coupon was moved to a second tube containing a biocide solution in the desired concentration, for an additional incubation of 1 hour .

The bromine-based biocide tested in this study was Bromosol® ( stabili zed Bromine , at 5 mg/L or 5 ppm) . Results

Figure 3 is a bar graph showing that Bromosol® at 5 mg/L ( 5 ppm) reduced the total count of bacteria in the biofilm by about 3 log units . Cis-2-undecenioc acid was shown to improve the ef ficacy of Bromosol® for biofilm removal , as demonstrated in Figure 3 . In particular, by adding cis-2-undecenioc acid at a concentration as low as 310 nM for 1 hour before the addition of the biocide , the total count reduction improved by additional 2 . 5 log units . In other words , in the presence of the combination of the biocide and cis-2-undecenioc acid, a total count reduction of about 5 log units as compared to the control was obtained .

Example 3 Enhancing the effect of a bromine-based biocide on massive biofilm with the aid of cis-2-nonenoic acid

The inventors have next examined the ef fect of cis-2-nonenoic acid ( CNA) in improving the ef ficacy of various biocides on biofilm removal and prevention, as detailed below .

Experimental procedure :

Speci fically, the single tube experiments were performed by first adding the fatty acids solution to the tube in the desired concentration for one hour . After one hour the coupon was moved to a second tube containing a biocide solution in the desired concentration, for an additional incubation of 1 hour . The bromine-based biocide tested in this study was Bromosol® ( stabili zed Bromine , at 5 mg/L or 5 ppm) .

Results

Figure 4 is a bar graph showing that Bromosol® at 5 mg/L ( 5 ppm) reduced the total count of bacteria in the biofilm by about 3 log units . Cis-2-nonenoic acid was shown to improve the ef ficacy of Bromosol® for biofilm removal , as shown in Figure 4 . In particular, by adding cis-2-nonenoic acid at a concentration as low as 310 nM for 1 hour before the addition of the biocide , the total count reduction improved by additional ~2 log units . In other words , in the presence of the combination of the biocide and cis-2-nonenoic acid, a total count reduction of about 4 . 5 log units as compared to the control was obtained .

Example 4 Enhancing the effect of a bromine-based biocide on massive biofilm with the aid of cis-2-octenoic acid

The inventors have next examined the ef fect of cis-2-octenoic acid ( COA) on improving the ef ficacy of various biocides in biofilm removal and prevention, as detailed below .

Experimental procedure :

The bromine-based biocide tested in this study was Bromosol® ( stabili zed Bromine , at 5 mg/L or 5 ppm) .

Results

Figure 5 is a bar graph showing that Bromosol® at 5 mg/L ( 5 ppm) reduced the total count of bacteria in the biofilm by about 3 log units . Cis-2-octenoic acid was shown to improve the ef ficacy of Bromosol® for biofilm removal , as demonstrated in Figure 5 . In particular, by adding cis-2-octenoic acid at a concentration as low as 310 nM for 1 hour before the addition of the biocide , the total count reduction improved by additional one and a hal f ( 1 . 5 ) log units . In other words , in the presence of the combination of the biocide and cis-2-octenoic acid, a total count reduction of about 4 log units as compared to the control was obtained .

Claims

34

Claims

1) A method of microbial control in water comprising adding to the water one or more bromine-based biocide (s) and at least one cis-2-alkenoic acid (CAA) of formula I,

where R is 63-6,8-15 alkyl, or a salt of said cis-2-alkenoic acid.

2) A method according to claim 1, wherein the microbial control comprises combatting planktonic bacteria and/or biofilm bacteria on a surface in contact with the water and/or inhibiting biofilm formation on a surface prone to biofilm growth.

3) A method according to claim 1 or claim 2, wherein the brominebased biocide is a non-oxidizing biocide.

4) A method according to claim 1 or claim 2, wherein the brominebased biocide is an oxidizing biocide.

5) A method according to claim 4, wherein the oxidizing biocide is a sulfamate-stabilized bromine-based biocide.

6) A method according to claim 5, wherein the sulfamate- stabilized bromine-based biocide is an aqueous solution of alkali hypobromite stabilized by sulfamic acid or a salt thereof.

7) A method according to claim 6, wherein the aqueous solution of alkali hypobromite stabilized by sulfamic acid or a salt thereof exhibits a strongly alkaline pH and has Br2 levels in the range from 15 to 18 %. 35

8) A method according to any one of the preceding claims, wherein low purity CAA(s) grade, of less than 95% purity as measured by gas chromatography (area %) , is added to the water.

9) A method according to any one of the preceding claims, wherein the bromine-based biocide (s) and CAA(s) are supplied to an industrial water stream in contact with an infested surface using multiple feed solutions, whereby the biocide (s) and CAA(s) are added sequentially or simultaneously to the water.

10) A method according to any one of the preceding claims, wherein the bromine-based biocide (s) and CAA(s) are supplied to an industrial water stream in contact with an infested surface using a single feed solution, whereby the biocide (s) and CAA(s) are added simultaneously to the water.

11) A method according to any one of the preceding claims, wherein the CAA is cis-2-heptenoic acid, cis-2-undecenoic acid, cis-2-nonenoic acid or cis-2-octenoic acid.

12) A method according to any one of the preceding claims, wherein the CAA is cis-2-heptenoic acid.

13) A method according to claim 11, wherein the bromine-based biocide is a sulfamate-stabilized based biocide and the CAA is cis-2-heptenoic acid.

14) A method according to any one of the preceding claims, wherein said method is for controlling Pseudomonas aeruginosa .

15) A composition comprising one or more bromine-based biocide (s) and cis-2-alkenoic acid(s) or salt(s) thereof of formula I as defined in claim 1.