Patent Description:
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 specific biocide under consideration, the dosing method, etc..

It has been reported by one of the present inventors [<CIT> and <NPL>)] that cis-<NUM>-decenoic acid, produced by the bacterium Pseudomonas aeruginosa, is capable of inducing P. aeruginosa and other gram-negative and gram-positive bacteria and 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 <CIT>, the performance of bromine-based biocides in controlling biofilms of P. aeruginosa was investigated. It has been also proposed in <CIT> to increase the efficiency of the treatment with the aid of surfactants that act as bio-dispersants, but no experimental data was given to illustrate this approach.

<CIT> relates to a method of controlling and removing biofilm on a surface in contact with an aqueous industrial system comprising the step of adding an effective amount of biofilm-disrupting agent and adding a biocide to the aqueous system being treated to reduce and remove biofilm-forming microbes from a surface in contact with the aqueous system.

The present invention describes the use of cis-<NUM>-decenoic acid as an adjunctive to bromine-containing biocides in the treatment of biofilm and planktonic bacteria in water systems and on surfaces in contact with the water. Experimental work conducted in support of this invention in laboratory models indicates that the combination of cis-<NUM>-decenoic acid with acceptable working concentrations of a bromine-containing biocide, shows a 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 biocides alone. Furthermore, the activity of cis-<NUM>-decenoic acid with the biocide compounds enhances the efficacy of brominated biocides, allowing for a reduction in the effective quantities of the biocides used.

It is also worth noting that while incorporation of cis-<NUM>-decenoic acid (CDA) into bromine-based water treatment greatly improves the effectiveness of biofilm control compared to the brominated biocide acting alone, a smaller effect is observed in chlorine-based water treatment. For example, it is shown below that under comparable conditions, the combined treatment bromine/CDA achieves biofilm bacteria count that is ~<NUM> log units lower than that achieved by chlorine/CDA treatment.

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) as defined in claim <NUM> and cis-<NUM>-decenoic acid (or a salt thereof) to achieve, for example, reduction of planktonic and/or biofilm bacteria, algae and fungi on a surface in contact with the water.

CDA 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 CDA 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 CDA and biocide 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.

To enable water treatment using a single additive feed instead of multiple additives feeds, we prepared liquid concentrates comprising suitably proportioned combinations of bromine-based biocide and CDA, which exhibit good room temperature storage stability.

Described herein is a composition (e.g., a liquid concentrate) comprising one or more bromine-based biocides and cis-<NUM>-decenoic acid 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 and CDA, e.g., granules, flakes & tablets, are also contemplated by the present invention.

Another aspect of the invention is a liquid concentrate as defined in claim <NUM>. A further aspect of the invention is a liquid concentrate as defined in claim <NUM>.

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 are commonly divided into two classes:.

Non-oxidizing biocides may be selected from the group of:.

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 Br- 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 CDA). The dosage of the oxidative biocides described herein is usually expressed as total Cl<NUM> that can be determined by iodometric titration using a titroprocessor: Titrino <NUM> plus or by DPD (Diethyl-p-PhenyleneDiamine) reagent method using a SQ-<NUM> spectrophotometer: Merck SQ-<NUM>. Oxidizing bromine-based biocides may be selected from the group of:.

Turning now to cis-<NUM>-decenoic acid, it can be used as pure oil dissolved in a suitable solvent, such as ethanol. High purity CDA, e.g., ≥<NUM>% pure by gas chromatography (GC), is commercially available from various sources such as Carbosynth Ltd. (Compton - Berkshire, United Kingdom) and Chemodex (St. Gallen, Switzerland). However, the experimental work reported below indicates that satisfactory enhancement of bromine-based water treatments can be achieved with the aid of CDA of lower purity, say, <NUM>%-<NUM>% pure oil by GC, e.g., <NUM>-<NUM>%, for example, <NUM>-<NUM>% (i.e., ~<NUM>%). The <<NUM>% (by gas chromatography, GC) pure CDA is named herein "low purity CDA grade". It may be appreciated that utilizing low purity CDA grade is economically advantageous. The term "pure CDA" refers to CDA characterized in having a purity level of more than <NUM>%, e.g., equal to or greater than <NUM>% as detected by GC.

CDA with any desired purity level can be obtained via the synthetic route described in <CIT>, by halogenating <NUM>-decanone CH<NUM>-(CH<NUM>)<NUM>-C(O)-CH<NUM>, to produce <NUM>,<NUM>-dihalide ketones (e.g., reaction with elemental bromine to produce <NUM>,<NUM>-dibromo-<NUM>-decanone), followed by dehalogenation in an alkaline environment generated by sodium or lithium hydroxide, to produce the terminal carboxylic acid group via the Favorski rearrangement and simultaneously the adjacent carbon-carbon double bond. The reaction mixture can then be worked-up by conventional techniques to recover CDA with purity levels suitable for use in the present invention, e.g. from <NUM> to <NUM>% (by GC).

The combination bromine/CDA has proved surprisingly effective against biofilm in laboratory models across a broad concentration range of various bromine-based biocides. Biofilm-associated bacterial counts measured for the combined treatment are ~<NUM>-<NUM> log units lower than for comparative values measured for the biocide acting alone. We use the term "enhancement" to indicate the difference in bacterial counts between treatments in which the biocide acts alone and in combination with CDA (CDA on its own does not reduce bacterial counts, as shown by the work reported below; notably, CDA alone failed to demonstrate biocidal action over a broad concentration range, even > <NUM>).

The performance of some selected bromine biocides, alone and in conjunction with different purity grades of CDA is tabulated in Table <NUM>. The results show the effect of bromine/CDA on <NUM>-day-old P. aeruginosa biofilm or biofilm formed by mixed bacteria, after short contact time: one hour contact time with CDA, followed by one hour contact time with the bromine-based biocide, at dosage levels of <NUM> and <NUM> - <NUM> ppm, respectively.

In another test reported below, the simultaneous application of one major non-oxidizing bromine biocide (bronopol) and CDA was proved effective in eradicating P. aeruginosa biofilm at the following treatment level: <NUM> ppm bronopol/<NUM> CDA after contact time of twenty-four hours. The results are tabulated in Table <NUM>.

Another significant set of results reported below shows that addition of a small amount of CDA can offset an appreciable decrease of the dosage level of the bromine-based biocide, thereby expanding the workable concentration range of bromine-based biocides. For example, with the aid of CDA, one major oxidizing biocide (bromourea) achieves reasonable biofilm control at a dosage level as low as <NUM> ppm (when acting alone at this dosage level, the biocide fails to generate a useful effect). The enhancement induced by the added CDA is roughly <NUM> log units. The same biocide eradicated biofilm at a dosage level of <NUM> ppm with <NUM> CDA. The results are summarized in Table <NUM>.

In view of the above, bromine-based water treatments could benefit from the addition of CDA in a number of ways:.

Accordingly, another aspect of the invention is a method of microbial control in water as defined in claim <NUM>, for example, reduction down to <<NUM><NUM> CFU/cm<NUM>, e.g., <<NUM><NUM> CFU/cm<NUM> and preferably <<NUM><NUM> CFU/cm<NUM> or even substantial biofilm eradication, i.e. <<NUM><NUM> CFU/cm<NUM>.

The effective microbiocidal amount of the bromine-based biocide(s) is from <NUM> to <NUM>, e.g., <NUM> to <NUM> ppm as active biocide, for example, <NUM> to <NUM> ppm, and the enhancement-inducing amount of CDA is from <NUM> to <NUM>. 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 : CDA as w/w in the water stream may vary in the range from <NUM>:<NUM> to <NUM>:<NUM> preferably from <NUM>:<NUM> to <NUM>:<NUM>. The enhancement-inducing amount of CDA can be determined by trial and error in the site of use to achieve targeted biofilm reduction.

For example, an enhancement-inducing amount of CDA could be from <NUM> to <NUM> ppm, e.g., from <NUM> to <NUM> ppm, for example, from <NUM> to <NUM> ppm. As shown below, good results were observed across <NUM> to <NUM> ppm (corresponding to ~<NUM> to <NUM> CDA).

<NUM>) Because cis-<NUM>-decenoic acid offsets reduction in bromine-based biocide dosing, allowing smaller quantities of the biocides to be used in an effective manner and achieve biofilm control comparable to higher dose treatments, cis-<NUM>-decenoic acid can modify program treatment by reducing biocide dosage level and/or frequency of biocide dosing. For example, the water system may be tracked for residual bromine and once the residual bromine values decay below a predetermined threshold, CDA can be injected to support the maintenance of the system with the low residual bromine to inhibit biofilm formation. That is, to enhance the activity of residual biocide in a water sample any time over the period of time that an active biocide is present in a system.

Described herein is a method of industrial water treatment comprising supplying bromine to the water for combatting biofilm bacteria on a surface in contact with the water and/or inhibiting biofilm formation on a surface prone to such formation, wherein the rate of application of bromine is varied over the treatment, such that switching to a low dosing level of bromine is accompanied by CDA addition to the water stream.

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.

<FIG> schematically illustrates one convenient method to feed a bromine-based biocide and CDA 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 (<NUM>). We use the term "industrial water" to indicate any aquatic industrial water treatable by a bromine-based biocide, 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 <FIG>, the biocide and CDA are held separately in tanks (<NUM>) and (<NUM>), 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 <FIG> 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., <NUM> to <NUM> wt% concentrates), and stabilized solutions of bromine or hypobromite (e.g., sulfamate-stabilized bromine-based biocide).

The design shown in <FIG> 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 CDA and two dosing pumps are used for the individual components of the biocide, i.e., the bromide source and the oxidant).

Incorporation of CDA 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 CDA 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 and CDA solutions are dosed with metering pumps (2p and 3p, respectively) controlled by timers set up according to the treatment program. The biocide and CDA feed solutions may be injected directly to the water stream (<NUM>) 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 (<NUM>) devices are included, namely, halogen monitoring, oxidation reduction potential (ORP), pH sensors and online static mixers.

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

In operation, sequential treatment with cis-<NUM>-decenoic acid can be performed by injecting the cis-<NUM>-decenoic acid from <NUM> minutes to <NUM> hours or more, prior to the biocide application. Cis-<NUM>-Decenoic acid 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 <FIG>. We have found that CDA is compatible with either the precursor of oxidative biocide (inorganic bromide sources), sulfamate-stabilized bromine-based biocides or with non-oxidizing biocides formulated in liquid concentrates, and that in the presence of suitable stabilizers, in particular an antioxidant (for example, butylated hydroxytoluene BHT), such liquid concentrates remain stable at room temperature over long storage periods against degradation of either the biocide or CDA. Accelerated tests also indicated acceptable stability. Such liquid concentrates can be dosed to the industrial water system using the proper feed system and are conveniently amenable to a simultaneous biocide/CDA treatment program. Accordingly, the invention also provides a method wherein the bromine-based biocide(s) and CDA are supplied to an industrial water stream in contact with an infested surface using a single feed solution, whereby the biocide and CDA are added simultaneously to the water. Described herein is a composition comprising one or more bromine-based biocides and cis-<NUM>-decenoic acid or a salt thereof (e.g., for use in the method).

For example, a nonoxidizing bromine-based biocide and CDA 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:.

One preferred room temperature storage stable liquid concentrate provided by the present invention comprises:.

Another preferred room temperature storage stable liquid concentrate provided by the present invention comprises:.

The concentrates are readily prepared by combining cis-<NUM>-decenoic acid, (or a salt thereof), 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.

Materials and reagents used in the experimental work are tabulated in Table <NUM>.

GC measurement was done with AGILENT HP-<NUM>19091J-<NUM><NUM>*<NUM>*<NUM> micron. Method: <NUM>//<NUM>'//<NUM>/min//<NUM>//<NUM>/min//<NUM>/<NUM>'.

Stock solution <NUM> (<NUM> of NaOCl <NUM>%) was added gradually while stirring to the above diluted Bactebrom® solution (stock solution <NUM>), to get the active biocide (orange solution) - total weight <NUM>. Expected biocide concentration as determined by iodometric titration using Titroprocessor: Titrino <NUM> plus. : ~ <NUM>% as Cl<NUM> (~<NUM> ppm as Cl<NUM>). The desired biocide concentration in each experiment was obtained by dilution with distilled water.

<NUM>µl <NUM> Wt% aq. NaOCl was diluted with distilled water to <NUM> in a volumetric flask. Cl<NUM> concentration was ~1000ppm as Cl<NUM> as determined by iodometric titration using Titroprocessor: Titrino <NUM> plus.

<NUM> NH<NUM>Br was diluted with distilled water to <NUM> in a volumetric flask.

Mix equal volumes of <NUM> as follows: add the NaOCl solution in one stroke to a mixed solution (using a magnetic stirrer) of the NH<NUM>Br solution at ambient temperature.

The concentration of the product (activated AmBr) was based on the concentration of the Na- Hypochlorite (~<NUM> ppm as Cl<NUM>).

Equal volumes of the reactants were mixed to obtain the concentration of the active chlorine in the mixture as <NUM>% of the concentration of the reactant NaOCl, ~<NUM> ppm as Cl<NUM>. The desired biocide concentration in each experiment was obtained by dilution with distilled water.

Cis-<NUM>-decenoic acid was prepared according to the two-step synthetic pathway described in <CIT>, replacing sodium hydroxide with lithium hydroxide in the second step (dehalogenation/rearrangement of <NUM>,<NUM>-dibromo-<NUM>-decanone) as depicted below:
<CHM>.

The reaction mixture was worked-up using conventional techniques (such as silica gel and solvent extraction) to recover cis-<NUM>-decenoic acid with purity varying in the range from <NUM> to <NUM>% (GC). In the studies reported below, <NUM>% pure cis-<NUM>-decenoic acid was tested.

The effect of bromine-containing biocide in combination with CDA on pre-grown biofilms was studied. The experiment was carried out utilizing (A) the P. aeruginosa strain PA14 and (B) mixed bacterial species derived from environmental and industrial water.

Three bromine-based biocides were tested in this study: bronopol, DBNPA and BCDMH.

Bacteria were cultured in EPRI medium supplemented with Hutners mineral solution and glucose (<NUM>%). Microorganisms were incubated at room temperature (<NUM>), under aerobic conditions with shaking. The biofilm culture system used included polystyrene <NUM>-well plates that were treated with protein to enhance attachment and growth of biofilm bacteria according to the method described in <NPL>.

Following inoculation with <NUM> bacterial culture, spent medium was removed and replaced with sterile medium every <NUM> hours for <NUM> days, and a final medium exchange was performed <NUM> hours prior to treatment. Additionally, the medium was exchanged prior to treatment in order to remove planktonic bacteria. Treatments consisted of <NUM>µL of <NUM> CDA and bromine-containing biocide, or bromine-containing biocide alone at concentrations used in commercial water treatment, water was used as a carrier, for a contact time determined by activity of each biocide and ranging from <NUM> hour to <NUM> hours, as detailed below for each of the tested biocides.

Following the treatment, the medium from each well was removed by pipet and <NUM> of DE neutralization broth was added in order to stop the treatment. Bacteria from each well were then removed by scraping the biofilm formed in the well with a sterile cell-scraper, and <NUM> of culture was transferred to chilled (<NUM>) <NUM> DE neutralizing broth and homogenized for <NUM> seconds at <NUM>,<NUM> rpm on ice. Further dilutions were performed prior to enumeration (further neutralizing biocide activity). Recovery of bacteria were tested at different dilutions of biocide in water, in LB medium with thioglycolate and in DE neutralizing broth to ensure the active agent was properly neutralized. Viable bacteria were enumerated via the drop plate method. Each biocide was evaluated using <NUM>-well plates with <NUM> wells each for the control cultures (Ctl) inoculated with P. aeruginosa but not treated, a CDA minus test (-CDA) treated with bromine-containing biocide only, and a CDA plus test treated with bromine-containing biocide and CDA (+CDA).

A) The results of the treatment of PA14 bacteria with <NUM> ppm of bronopol are presented in <FIG> shows that the addition of CDA to the treatment with bronopol reduced the number of bacteria by more than an order of magnitude after only two hours of treatment. <FIG> demonstrates the effect achieved after <NUM> hours of treatment. It is seen that bronopol alone is very effective in reducing biofilm. Nevertheless, with the aid of added CDA, the treatment became much more effective and led to biofilm eradication.

The results of the treatment of PA14 bacteria with <NUM> ppm of DBNPA are presented in <FIG> in the form of a bar diagram. It is seen that a short treatment of two hours which included both DBNPA and CDA yielded an improved effect compared with a treatment which was carried under the same conditions with DBNPA alone.

The results of the treatment of PA14 bacteria with <NUM> ppm of BCDMH are presented in <FIG> in the form of a bar diagram. It is seen that a short treatment of just one hour, which included the application of both BCDMH and CDA, generated an improved result compared with treatment using BCDMH alone (log reduction = <NUM>).

The results of the treatment against undefined mixed microbial biofilm cultures derived from an environmental water source and against a biofilm culture derived from fresh-water mixed microbial community that were obtained from cooling tower water, with <NUM> ppm of DBNPA are presented in <FIG> respectively. It is seen that a short treatment of two hours which included both DBNPA and CDA yielded an improved result, compared with a treatment which was carried out under the same conditions with DBNPA alone.

The effect of the sequential addition of CDA and bromine-containing biocide was 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 took place <NUM> after the addition of <NUM> CDA. See Sections A-F, corresponding to six bromine-based biocides that were tested. In section G herein below, two more bacteria strains (staphylococcus aureus <NUM>, bacillus mycoides <NUM>) were added to the reactor and contributed to the formation of a mixed biofilm. In section H herein below, two other CDA concentrations (<NUM> and <NUM>) were tested against P. aeruginosa biofilm.

Six bromine-based biocides were tested (DBNPA, sulfamate-stabilized bromine, BCDMH, bromourea, activated ammonium bromide and activated NaBr) in combination with two CDA products of different purity: commercial CDA (CV-CHEM, <NUM>% by GC) and crude CDA (<NUM>% by GC) of Preparation <NUM>, to evaluate the effect of the purity level of the CDA used.

The efficacy test on the coupons was performed according to the single tube method (E2871-<NUM>). This test method is used for growing a reproducible P. aeruginosa biofilm in a CDC Biofilm Reactor.

The biofilm was established by operating the reactor in batch mode (no flow of the nutrients) for <NUM>. A steady state population was reached after the reactor operated for an additional <NUM> days with continuous flow of the nutrients. During the entire <NUM>-day period, the biofilm was exposed to continuous fluid shear from the rotation of a baffled stir bar. At the end of the <NUM> days, the biofilm from the coupons was sampled as follows:.

Sequential addition of CDA and bromine-containing biocide:.

The goal of the study was to estimate to which extent the addition of CDA can offset a decrease of the dosage level of the bromine-based biocide. That is, to offer combined bromine/CDA treatment program that is equally effective as currently acceptable, high-dosage level, acting-alone bromine.

The biocide tested was BCDMH. CDA of commercial source, <NUM>% purity grade, was used. The experimental protocol of Example <NUM> was repeated (i.e., sequential treatment, CDA followed by BCDMH, applied to biofilm grown for three days, <NUM> hour biocide contact time).

The results are presented in the form of a bar diagram in <FIG>, showing that the effect of <NUM> ppm of BCDMH in the presence of <NUM> CDA is comparable to that of <NUM> ppm BCDMH in the absence of CDA. Hence, addition of very small amount of CDA to the water can offset a <NUM>% decrease in the dosage level of the biocide.

CDA (commercial <NUM>% purity grade) was applied in combination with varying quantities of bromine-based biocide (the dosage level of the biocide was varied in the range from <NUM> to <NUM> ppm) to investigate the ability of CDA to support the action of the biocide across a wide biocide concentration range. CDA was used at a constant concentration of <NUM>. The combined treatment bromine/CDA was compared to the solely applied bromine-based biocide.

The biocide tested was bromourea. The experimental protocol of Example <NUM> was repeated (i.e., sequential treatment, CDA followed by bromourea, applied to biofilm grown for three days, <NUM> hour biocide contact time).

The results are shown in <FIG>, in the form of survival plots corresponding to the solely applied biocide (marked by rhombuses) and the combined biocide/CDA treatment. The concentrations of the biocide tested were <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> ppm. The results indicate the effectiveness of the combined treatment against <NUM>-day biofilm over the entire biocide concentration range (i.e., ><NUM> ppm), demonstrating LC<NUM> (biofilm concentration at which <NUM>% of the culture is killed) biofilm control at treatment level of <NUM> ppm bromourea/<NUM> CDA and biofilm eradication concentration as low as <NUM> ppm bromourea/<NUM> CDA.

<NUM> CDA and bromine-containing biocide were introduced into a mix of planktonic bacteria in a medium containing different levels of organic loading (TOC <NUM>-<NUM> ppm). High organic loading tends to reduce the efficacy of antimicrobial agents in industrial applications and, therefore, the test was aimed to mimic these conditions. The test was performed in accordance with the modified European standard EN <NUM>: <NUM>: "Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics - Tests method and requirements (phase <NUM>)". <NUM> of phosphate buffer solution (pH=<NUM>) including tryptone in order to obtain a solution of TOC=<NUM> ppm, and <NUM> of the tested bacteria suspension (consisted of E. Coli (ATCC <NUM>), S. aureus (ATCC <NUM>), Entrobacter aerogenes (ATCC <NUM>) and P. aeruginosa (ATCC <NUM>)), at a concentration of <NUM>×<NUM><NUM>-<NUM>×<NUM><NUM> CFU/ml, were placed into a container of suitable capacity. A stopwatch was started immediately and the container was placed in a water bath controlled at <NUM>.

The activity was determined with <NUM> ppm BCDMH alone for a contact time of <NUM> hours. The combination of CDA (CV-CHEM) with <NUM> ppm BCDMH was tested when added in sequence (<NUM> hour with CDA then additional <NUM> hours with BCDMH). At the desired contact time, <NUM> of the tested mixture was pipetted into a tube containing <NUM> neutralizer. Immediately after <NUM> sec of neutralization time, a sample of <NUM> was taken in duplicate and transferred to a Petri dish. TSA, cooled to <NUM>±<NUM>, was added. The plates were incubated at <NUM>±<NUM> for <NUM> hours. Countable plates were counted and the number of colony-forming units was determined, for each plate.

The results are presented in <FIG>. As can be seen, enhancement of <NUM> log units was obtained when CDA (CV-CHEM) and BCDMH (<NUM> ppm) were introduced in sequence compared with the effect achieved by BCDMH in the absence of CDA.

<NUM> of cis-<NUM>-decenoic acid (CiVentiChem, <NUM>% purity) were charged into <NUM> flask, equipped with a stirrer. <NUM> gr of C-<NUM> (ICL, batch <NUM>) were added, followed by <NUM> gr of PEG200 (Merck <NUM>. <NUM> Lot S6904983 <NUM>), <NUM> gr of DI water and <NUM> of BHT (Aldrich B1378-<NUM>, BCBH9491V). The obtained solution was stirred till complete clarification; sonication at +<NUM> is recommended if some particles are observed. Clear colorless solution of a total <NUM> gr and <NUM>:<NUM>:<NUM>:<NUM>:<NUM> weight ratio of C103-PEG<NUM>-H<NUM>O-CDA-BHT was obtained.

The solution was tested to determine the stability of the biocide and CDA and under storage at <NUM> over a two-month period. Neither DBNPA nor CDA underwent degradation in the <NUM>/two-month test (analysis by HPLC).

<NUM> of cis-<NUM>-decenoic acid (CiVentiChem, <NUM>% purity) were charged into <NUM> flask, equipped with a stirrer. <NUM> gr of Bronopol (ICL, batch <NUM>) were added, followed by <NUM> gr of propylene glycol (Biolab <NUM> Lot <NUM>), <NUM> gr of DI water and <NUM> of BHT (Aldrich B1378-<NUM>, BCBH9491V). After stirring for several minutes, undissolved traces of BHT were filtered through filtering paper for BHT particle removal. Clear colorless solution of a total <NUM> gr and <NUM>:<NUM>:<NUM>:<NUM>:<NUM> weight ratio of Bronopol-PG-H<NUM>O-CDA-BHT was obtained.

The purpose of the set of experiments reported in this Example was to check if the addition of CDA to bromine- and chlorine-based treatments generate comparable effects on the targeted biofilm. That is, whether CDA augments the action of bromine and chlorine on biofilm in equally effective manner. Sodium hypochlorite and BCDMH were chosen as illustrative chlorine and bromine biocides, respectively.

CDA of commercial source, <NUM>% purity grade, was used. The experimental protocol of Example <NUM> was repeated (i.e., sequential treatment, CDA followed by the halogenated biocide, applied to biofilm grown for three days, <NUM> hour biocide contact time).

The bar diagram of <FIG> shows that sodium hypochlorite and BCDMH applied alone at dosage level of <NUM> ppm have comparable effect on biofilm. However, a surprisingly better effect was generated by the bromine/CDA combination of the invention on biofilm, compared with chlorine/CDA. The combination BCDMH/CDA applied at treatment level of <NUM> ppm BCDMH/<NUM> CDA achieved <NUM> log units reduction versus the solely applied BCDMH treatment, whereas the corresponding hypochlorite/CDA treatment was able to improve to a lesser extent (<NUM> log units reduction).

The effect of the sequential addition of CDA and bromine-containing biocide was studied using P. aeruginosa biofilm grown in the drip flow tubes (according to a modification of standard ASTM <NUM>-<NUM>, standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown Using Drip Flow Biofilm Reactor with Low Shear and Continuous Flow). This method produces massive biofilm resulting with harsh conditions that are very difficult to treat. The addition of the bromine-containing biocide took place <NUM> after the addition of <NUM> CDA (the biocide that was tested in this study was DBNPA).

The results presented in <FIG> show that the effect of <NUM> ppm of DBNPA in the combined treatment of <NUM> CDA enhanced the biocidal effect of the biocide alone by ca <NUM> log orders.

Experimental stepwise acidification of CDA sodium/lithium salt indicates that cis-DA pKa value is in the range of <NUM>-<NUM>. In several industrial applications the pH values of the treated water have higher alkalinity and therefore will promote the formation of the salt form of cis-DA. The effect of the simultaneous addition of CDA in its salt form and bromine-containing biocide was studied using P. aeruginosa biofilm grown in the CDC biofilm reactor on borosilicate glass coupons (test method E2562). The bromine-containing biocide tested was sulfamate-stabilized bromine (Bromosol), added simultaneously with <NUM> CDA for <NUM>. The pH of the treated solution was in the range of pH <NUM>-<NUM> - conditions which promote the formation of the salt of cis-DA.

Simultaneous addition of CDA and bromine-containing biocide:.

After the one-hour contact time, <NUM> of a neutralizer was added to each tube.

Claim 1:
A method of microbial control in water comprising adding to the water one or more bromine-based biocide(s) and cis-<NUM>-decenoic acid or a salt thereof, wherein said bromine-based biocide is selected from:
A) a non-oxidizing bromine-based biocide selected from the group consisting of:
A1)<NUM>-bromo-<NUM>-nitro-<NUM>,<NUM>-propanediol (Bronopol) and
A2) <NUM>,<NUM>-dibromo-<NUM>-nitrilopropionamide (DBNPA);
B) an oxidizing bromine-based biocide selected from the group consisting of:
B1) <NUM>-bromo-<NUM>-chloro-<NUM>,<NUM>-dimethylhydantoin (BCDMH);
B2) an on-site oxidized bromide source, which releases active bromine species in water; and wherein the on-site oxidized bromide source is selected from:
sodium bromide, which is oxidized on-site with hypochlorite, chlorine or electrochemically to produce its active form, to be added to the water system to be treated;
HBr, which is oxidized on-site with hypochlorite, chlorine or electrochemically to produce its active form, to be added to the water system to be treated;
ammonium bromide, which is oxidized on-site with hypochlorite, chlorine or electrochemically to produce its active form, to be added to the water system to be treated;
solution of HBr/NaBr and urea, which reacts with hypochlorite, chlorine or electrochemically on-site to produce the bromourea active form, to be added to the water system to be treated; and
B3) sulfamate stabilized aqueous solution of alkali hypobromites (bromosulfamate).