Method for controlling growth of microorganisms and/or biofilms in an industrial process

Abstract

Disclosed is a method for controlling a biofilm removing a formed biofilm and/or controlling a growth of microorganisms, preferably bacteria, in an aqueous environment of an industrial manufacturing process including a cellulosic fibre material. A compound according to Formula I is administered to the aqueous environment, in which Formula I R1, R2 and R3 independently represent a hydrogen atom; halogen atom; hydroxy group; amino group; alkylamino group, alkyl group, hydroxyalkyl group, haloalkyl group or alkoxy group having 1 to 4 carbon atoms; or an acylamido group having 1 to 10 carbon atoms; and A represents 2-thiazolamine; 2-propenenitrile; 2-propenoic acid; alkyl ester or hydroxyalkyl ester of 2-propenoic acid having 1 to 4 carbon atoms; or —CHCHCONR5R6 group, where R5 and R6 represent independently hydrogen atom, alkyl or hydroxyalkyl having 1 to 4 carbon atoms, with the proviso that the compound according to Formula I is not 3-[(4-methylphenyl)sulphonyl]-2-propenenitrile or 4-amino-N-2-thiazolyl-benzenesulphonamide.

Claims

1. A method for controlling a biofilm, for removing a formed biofilm and/or for controlling a growth of microorganisms in an aqueous environment of an industrial manufacturing process comprising a cellulosic fibre material and having a temperature of at least 40° C. and being selected from manufacture of paper, board, pulp, tissue, moulded pulp, or non-woven, the method comprising: adding continuously or periodically into the aqueous environment, a composition comprising a compound according to ##STR00002## wherein R1, R2 and R3 independently represent a hydrogen atom; halogen atom; hydroxy group; amino group; alkylamino group, alkyl group, hydroxyalkyl group, haloalkyl group or alkoxy group having 1 to 4 carbon atoms; or an acylamido group having 1 to 10 carbon atoms; and A represents 2-thiazolamine; 2-propenenitrile; 2-propenoic acid; alkyl ester or hydroxyalkyl ester of 2-propenoic acid having 1 to 4 carbon atoms; or —CHCHCONR5R6 group, where R5 and R6 represent independently hydrogen atom, alkyl or hydroxyalkyl having 1 to 4 carbon atoms, with the proviso that said compound is not 3-[(4-methylphenyl)sulphonyl]-2-propenenitrile or 4-amino-N-2-thiazolyl-benzenesulphonamide.

2. The method according to claim 1, wherein in Formula (I): R1 represents a methyl group; ethyl propyl group; butyl group; methoxy group; ethoxy group; propoxy group; isopropoxy group; n-butoxy group; or tertiary butoxy group; and R2 and R3 represent independently a hydrogen atom; methyl group; ethyl propyl group; butyl group; methoxy group; ethoxy group; propoxy group; isopropoxy group; n-butoxy group; tertiary butoxy group; and A represents 2-propenenitrile; R1, R2, R3 being located independently in ortho, meta or para position relative to A.

3. The method according to claim 1, wherein in Formula (I): R1 represents a methyl group; ethyl propyl group; butyl group; methoxy group; ethoxy group; propoxy group; isopropoxy group; n-butoxy group; tertiary butoxy group; or amino group; and R2 and R3 represent independently a hydrogen atom; methyl group; ethyl propyl group; butyl group; methoxy group; ethoxy group; propoxy group; isopropoxy group; n-butoxy group; tertiary butoxy group; and A represents —CHCHCONR5R6 group, where R5 and R6 represent independently hydrogen atom; alkyl or hydroxyalkyl having 1 to 4 carbon atoms; R1, R2, R3 being located independently in ortho, meta or para position relative to A.

4. The method according to claim 3, wherein R5 and R6 in —CHCHCONR5R6 group represent hydrogen atoms.

5. The method according to claim 1, wherein the compound according to Formula (I) is selected from group consisting of 3-phenylsulphonyl-2-propenenitrile, 3-[(4-fluorophenyl)sulphonyl]-2-propenenitrile, 3-[(4-trifluormethylphenyl)sulphonyl]-2-propenenitrile, 3-[(2,4-dimethylphenyl)sulphonyl]-2-propenenitrile, 3-[(3,4-dimethylphenyl)-sulphonyl]2-propenenitrile, 3-(3,5-dimethylphenyl)sulphonyl-2-propenenitrile, 3-[(2,4, 6-trimethylphenyl)sulphonyl]-2-propenenitrile, 3-(4-methoxyphenyl)sulphonyl-2-propenenitrile, 3-[(4-methylphenyl)sulphonyl]prop-2-enamide, 3-[(4-methylphenyl)sulphonyl]prop-2-enoic acid, and any of their isomers.

6. The method according to claim 5, wherein the compound according to Formula (I) is selected from group consisting of 3-phenylsulphonyl-2-propenenitrile, 3-[(4-trifluormethylphenyl)sulphonyl]-2-propenenitrile, 3-[(2,4, 6-trimethylphenyl)sulphonyl]-2-propenenitrile, 3-(4-methoxyphenyl)sulphonyl-2-propenenitrile and 3-[(4-methylphenyl)-sulphonyl]prop-2-enamide; and any of their isomers.

7. The method according to claim 1, wherein the composition is administered to the aqueous environment in amount of 0.01-100 ppm, calculated as active compound.

8. The method according to claim 7, wherein the composition is administered in amount of 0.01-10 ppm calculated as active compound.

9. The method according to claim 8, wherein the composition is administered in amount of 0.01-2 ppm, calculated as active compound.

10. The method according to claim 1, wherein the composition is administered to the aqueous environment in amount of 0.01-1 ppm, calculated as active compound.

11. The method according to claim 10, wherein the composition is administered to the aqueous environment in amount of 0.01-0.5 ppm, calculated as active compound.

12. The method according to claim 11, wherein the composition is administered to the aqueous environment in amount of 0.01-0.3 ppm, calculated as active compound.

13. The method according to claim 1, wherein the aqueous environment comprises bacteria belonging to genus of Meiothermus, Deinococcus and/or Pseudoxanthomonas, either alone or in any combination or the aqueous environment is in contact with a biofilm at least partially formed by any of said bacteria.

14. The method according to claim 1, wherein the aqueous environment comprises water; cellulosic fibres; and further optionally starch; inorganic mineral particles; hemicelluloses; lignin; and/or dissolved and colloidal substances.

15. The method according to claim 14, wherein the cellulosic fibres are lignocellulosic fibres and inorganic mineral particles are selected from fillers and coating minerals.

16. The method according to claim 1, wherein the composition is administered to the aqueous environment, which comprises a residual of peroxide from about 0.01 to about 100 ppm.

17. The method according to claim 1, wherein the temperature of the aqueous environment is at least 50° C.

18. The method according to claim 1, wherein the composition is administered periodically in the aqueous environment for 3-45 minutes for 6-24 times a day.

19. The method according to claim 18, wherein the composition is administered periodically for 10-30 minutes for 12-24 times a day.

20. The method according to claim 1, wherein the composition is used in addition with other biocidal or antimicrobial agents.

21. The method according to claim 20, wherein the composition is administered to the aqueous environment, which comprises a residual of active halogen in the range from about 0.01 to about 20 ppm, given as active chlorine.

22. The method according to claim 1, wherein microorganisms are bacteria.

Description

EXPERIMENTAL

(1) Some embodiments of the invention are described more closely in the following non-limiting examples.

(2) Materials and Methods Used in the Examples

(3) Pure cultures of Meiothermus silvanus, a microbe species commonly found in paper machine biofilms (Ekman J, Journal of Industrial Microbiology & Biotechnology 34:203-211) and Pseudoxanthomonas taiwanensis, another species commonly found in paper machine environments (Desjardins, E & Beaulieu, C, Journal of Industrial Microbiology & Biotechnology 30:141-145) were used to study the efficacy of various chemicals to prevent biofilm formation.

(4) Biofilm tests were done in either synthetic commercial R2-broth (Lab M Ltd, UK) or fibre-containing synthetic paper machine water, SPW (prepared according to Peltola, et al., J. Ind. Microbiol. Biotechnol. 2011, 38: 1719-1727) using 96-microwell plate wells with peg lids (Thermo Fischer Scientific Inc., USA). Plates were incubated at 45° C. with a rotary shaking (150 rpm) providing high flow in each well.

(5) 2,2-dibromo-3-nitrilopropionamide, hereinafter called DBNPA, was obtained from Kemira Oyj (Fennosan R20, 20% active ingredient).

(6) (2E)-3-phenylsulphonyl-2-propenenitrile, hereinafter called Compound C was synthesised as follows:

(7) 1.066 g (0.00533 mol) of C.sub.6H.sub.5SO.sub.2Na×2H.sub.2O was weighed into 50 ml flask. 3 ml H.sub.2O and 1 ml AcOH were added followed by stirring until complete dissolution. 0.466 g (0.00533 mol, 1 eq.) of 2-chloroacrylonitrile was added to the clear solution. Mixture was stirred for 0.5 h, followed by addition of 7 ml of H.sub.2O and extra 15 minutes of stirring. The mixture was left to refrigerator overnight. After that mixture was filtered, washed with 50 ml cold water and dried on lyophilizer. Mass of the intermediate product was 0.900 g (yield 73.6%). The intermediate product was transferred into 100 ml flask and dissolved in 60 ml of MTBE. 0.396 g of Et.sub.3N was added dropwise instantly forming white precipitate in the solution. Reaction mixture was stirred 1 h. After that, the mixture was filtered and the residue was washed with 20 ml MTBE. Filtrate was extracted with 2×50 ml 1 M KHSO.sub.4 solution. After that, organic phase was evaporated under reduced pressure and the residue was dried on lyophilizer.

(8) Mass of the product was 0.667 g (yield 88.1%). Purity (HPLC): 99.1%.

(9) .sup.1H NMR: (CDCl.sub.3, 700 MHz) δ 7.96-7.87 (m, 2H), 7.74 (s, 1H), 7.63 (s, 2H), 7.24 (d, J=15.7 Hz, 1H), 6.55 (d, J=15.7 Hz, 1H).

(10) (2E)-3-[(2,4,6-trimethylphenyl)sulphonyl]-2-propenenitrile, hereinafter called Compound D, was synthesised as follows:

(11) 300 mg (1.45 mmol) sodium 2,4,6-trimethylphenyl sulphinate was dissolved in mixture of 0.18 ml acetic acid and 0.5 ml of water. 115 μl (126 mg, 1.44 mmol) 2-chloroacrylonitrile was added and the mixture was stirred for 1 h at room temperature. After this time, 0.5 ml of water was added and the mixture was stirred for additional 20 minutes. The product precipitated as oil (very slightly yellow), it could not be separated by filtration. The reaction mixture was neutralised to pH 6.8 using saturated solution of NaHCO.sub.3 and extracted with MTBE (4×3 ml).

(12) Combined MTBE fractions were analysed and 221 μl (160 mg, 1.58 mmol, 1.1 eq) trimethylamine was added. The mixture was stirred for 1 h at room temperature, extracted twice with 1M KHSO.sub.4 (3 ml) and once with sat. NaCl solution (5 ml), dried over Na.sub.2SO.sub.4 and evaporated to dryness. 60 mg of slightly yellow solid was obtained (yield 17% over 2 steps). The identity of the product was confirmed by 1H and 13C NMR, purity of the product was 94.5% by HPLC.

(13) The reason for low yield was found to be incomplete conversion in the first step—the aqueous phase after extraction of the intermediate with MTBE contained high concentration of the starting sodium 2,4,6-trimethylphenyl sulphonate.

(14) .sup.1H NMR: (CDCl.sub.3, 700M Hz) δ 7.27 (d, J=15.7 Hz, 1H), 7.02 (d, J=0.5 Hz, 2H), 6.45 (d, J=15.7 Hz, 1H), 2.60 (s, 6H), 2.33 (s, 3H).

(15) .sup.13C NMR (176 MHz, CDCl.sub.3) b 149.54, 145.29, 140.86, 132.77, 130.45, 113.61, 108.96, 22.89, 21.16.

(16) (2E)-3-[(4-trifluormethylphenyl)sulphonyl]-2-propenenitrile, hereinafter called Compound E, was synthesised as follows:

(17) 300 mg (1.29 mmol) sodium 4-trifluoromethylphenyl sulphinate was dissolved in mixture of 0.16 ml acetic acid and 0.44 ml of water. 103 μl (112 mg, 1.28 mmol) 2-chloroacrylonitrile was added and the mixture was stirred for 1 h at room temperature. After this time, 0.5 ml of water was added and the mixture was stirred for additional 20 minutes. The product precipitated as amorphous, orange solid, it could not be separated by filtration. The reaction mixture was neutralised to pH 6.8 using saturated solution of NaHCO.sub.3 and extracted with MTBE (4×3 ml).

(18) MTBE fractions were combined and 195 μl (142 mg, 1.4 mmol, 1.1 eq) trimethylamine was added. The mixture was stirred for 1 h at room temperature, extracted twice with 1M KHSO.sub.4 (3 ml) and once with sat. NaCl solution (5 ml), dried over Na.sub.2SO.sub.4 and evaporated volume ˜1 ml. Product precipitated as light crystals, the mother liquor was orange. 53 mg of slightly yellow solid was obtained (yield 16% over 2 steps). The identity of the product was confirmed by 1H and 13C NMR, purity of the product was 88.4% by HPLC. The reason for low yield was here likely also low conversion in first step and also incomplete precipitation of product from MTBE (which was needed, as the mother liquor was clearly coloured).

(19) .sup.1H NMR (700 MHz, CDCl.sub.3) δ 8.06 (d, J=8.2 Hz, 2H), 7.90 (d, J=8.3 Hz, 2H), 7.23 (d, J=15.6 Hz, 1H), 6.63 (d, J=15.6 Hz, 1H).

(20) .sup.13C NMR (176 MHz, CDCl.sub.3) δ 148.09 (s), 140.96 (s), 136.68 (q, J=33.4 Hz), 129.19 (s), 127.11 (q, J=3.6 Hz), 122.84 (d, J=273.4 Hz), 112.97 (s), 112.18 (s).

(21) (2E)-3-[(4-methoxyphenyl)sulphonyl]-2-propenenitrile, hereinafter called Compound F, was synthesised as follows:

(22) 1.257 g (0.00998 mol, 2 eq.) of Na.sub.2SO.sub.3 and 0.846 g (0.01007 mol, 2 eq) of NaHCO.sub.3 was weighted into 50 ml flask and dissolved in 30 ml of H.sub.2O/THF 10:1. The solution was cooled down in ice bath and 1.064 (0.00515 mol, 1 eq.) of para-methoxyphenylsulphonyl chloride was added dropwise during 5 min. Reaction mixture was stirred for 3 hours at room temperature. After that, the clear reaction mixture was extracted with 3×20 ml CHCl.sub.3. Water phase was evaporated under reduced pressure and the residue was stirred with 2×25 ml of MeOH followed by filtration. The solid inorganic residue was removed and the filtrate was evaporated under reduced pressure. Circa 4 g of white solid material was obtained (yield over 100% due to presence of inorganic components). This material was used in the next step without further treatment.

(23) The material was transferred into 50 ml flask and dissolved in the mixture of 7 ml H.sub.2O and 2.8 ml AcOH. After that, 0.410 ml (0.451 g, 0.00515 mol) of 2-chloroacrylonitrile was added dropwise. The mixture was stirred for 50 minutes before 4 ml H.sub.2O was added followed by extra 15 minutes of stirring. Clear oil-like substance precipitated out. pH of the reaction mixture was increased to 6.85 with saturated NaHCO.sub.3 solution; oil was dissolved. Reaction mixture was extracted with 3×25 ml MTBE and organic phases were transferred into 250 ml flask. This solution was used directly in next step without further treatment.

(24) 0.720 ml (0.523 g, 0.00517 mol) of Et3N was added to the obtained solution. The reaction mixture was stirred for 1 h. Reaction mixture was washed with 2×50 ml of 1 M KHSO.sub.4 solution and 10 ml of saturated NaCl solution. Organic phase was evaporated under reduced pressure and dried on lyophilizer. The mass of the final product was 0.639 g (yield 55% over 3 steps). HPLC purity 93.5%. Identity of compound 1 K was confirmed with NMR.

(25) .sup.1H NMR (700 MHz, CDCl.sub.3) δ ppm 3.92 (s, 3H) 6.50 (d, J=15.61 Hz, 1H) 7.08 (d, J=9.05 Hz, 2H) 7.23 (d, J=15.61 Hz, 1H) 7.83 (d, J=9.06 Hz, 2H)

(26) .sup.13C NMR (176 MHz, CDCl.sub.3) δ ppm 55.92 (s) 109.53 (s) 113.53 (s) 115.24 (s) 128.32 (s) 130.92 (s) 149.59 (s) 164.93 (s)

(27) (2E)-3-[(4-methylphenyl)sulphonyl]prop-2-enamide, hereinafter called Compound G, was synthesised as follows:

(28) 3.26 g (0.0469 mol) NH.sub.2OH×HCl was dissolved in 50 ml of NaOH (1M)/THF 1:1 in 100 ml flask. The solution was cooled down in ice bath and 2.54 g (0.0630 mol, 1.3 eq.) of acetaldehyde was added. The mixture was stirred at room temperature for 6 hours. pH of the reaction mixture was around 1. After the reaction, the solution was neutralized with 2 M NaOH. As it was desired to reduce the amount of THF in the mixture for next synthesis step, the mixture was evaporated a bit under vacuum at 40° C. After 5 min of evaporation (around 10 ml evaporated), colour of the solution turned slightly pink and evaporation was stopped. The mixture was left overnight into refrigerator.

(29) 1.009 g (0.00487 mol) of nitrile was added to the prepared mixture. Around 100 mg of NiCl2×6H.sub.2O was added to the mixture as catalyst and the reaction mixture was heated to reflux. Formation of the product was monitored with TLC (PE/EA 5:1).

(30) After 2 hours, the reaction mixture had brown colour, TLC showed that almost all nitrile had already reacted. As the product didn't move on TLC with solvent PE/EA 5:1, new solvent CHCl.sub.3/MeOH 10:1 was used instead. This indicated that after 4 hours of reflux, nitrile wasn't anymore present in the solution, also pure product spot without any significant impurities were notified. Reflux was ended and the mixture was cooled to room temperature. After further cooling in ice bath, mixture was filtered and washed with 100 ml of water. Light gray product was left to dry on lyophilizer overnight.

(31) Mass of the product was 0.461 g. For further purification, column chromatography with eluent CHCl.sub.3/MeOH 10:1 was performed. 50 g of medium size silica gel was used and 18 fractions (50 ml each) were collected. Each fraction was analysed with TLC (CHCl.sub.3/MeOH 10:1). In the first fraction, there were impurities and therefore fractions 2-9 were collected. Fractions 10-18 didn't contain significant amount of the product. Solution (fractions 2-9) was evaporated under vacuum and dried in lyophilizer. Mass of the final product was 0.317 g. HPLC Purity: 94.4%.

(32) .sup.1H NMR (700 MHz, DMSO) δ 8.02 (s, 1H), 7.82-7.78 (m, 2H), 7.67 (s, 1H), 7.50-7.47 (m, 2H), 7.41 (d, J=15.0 Hz, 1H), 6.95 (d, J=15.0 Hz, 1H), 2.41 (s, 3H). .sup.13C NMR (176 MHz, DMSO) δ 163.13, 145.07, 139.28, 135.95, 134.80, 130.25, 127.75, 21.11.

(33) Test Method for Prevention of Biofilm Formation

(34) For experiments of preventing biofilm formation wells of 96-microwell plates with peg-lids were filled with R2-broth or SPW, inoculated with the pure bacterial cultures and treated with different amounts of chemical compounds to be tested. Peg-lid was put on. After 24 hours the wells were emptied and a fresh solution of pure culture containing SPW or R2 broth with different amounts of test chemicals were added to the wells and the original peg-lid was put back in place. After an additional 24 hours, i.e. 48 hours after starting the test, the wells were emptied, rinsed and the peg lid and wells were left to dry.

(35) Test Method for Removal of Existing Biofilm

(36) For experiments of removing already existing (preformed) biofilm wells of 96-microwell plates with peg-lids were filled with SPW, inoculated with the pure bacterial cultures. Biofilm was grown for 24 hours without addition of any chemical compound to be tested. In some experiments after 24 hours the procedure was repeated by emptying the wells and by addition of a fresh solution of SPW inoculated with pure bacterial culture, again without any test chemical compound. The original peg-lid was put back in place and biofilm was allowed to grow for additional 24 h, i.e. in total 48 h.

(37) After 24 or 48 hours after starting the test, the wells were emptied and a fresh solution of SPW, inoculated with the pure bacterial cultures and with different amounts of chemical compounds to be tested were added and the original peg-lid was placed back in place. After an additional 2 or 24 hours the wells were emptied, rinsed and the peg lid and wells were left to dry.

(38) Quantification of Formed Biofilm

(39) The amount of biofilm formed on the microwells and peg surfaces was quantified with a staining solution by adding 200 μl of 1% Crystal Violet (Merck Millipore KGaA, Germany) in methanol to each well and placing the peg-lid back on. After 3 minutes the wells were emptied and the wells and pegs were rinsed 3 times with tap water. The attached Crystal Violet was dissolved into ethanol and the absorbance at 595 nm was measured. The values shown in the following tables are average absorbance from 8 replicate wells and pegs.

(40) All absorbance values in Examples 1-8 are given actual measured values. In calculation for biofilm reduction percentages it was taken in account that the SPW alone for 2 days without any bacterial inoculum gave a background value of 0.14.

Example 1 (Reference)

(41) Tables 1 and 2 demonstrate the ability of a conventional antimicrobial agent DBNPA to prevent biofilm formation of Meiothermus silvanus and Pseudoxanthomonas taiwanensis. Test conditions simulated paper or board making process conditions (synthetic paper machine water, high temperature, fibres present, high flow). The conventional antimicrobial agent DBNPA required a dosage of 1 mg/I active compound to reach acceptable or noticable biofilm reduction efficacy. The results for DBNPA are given in Tables 1 and 2.

(42) Table 1 shows the effect of DPNPA dosing to Meiothermus silvanus biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was stained and quantified by absorbance measurement. Dosage given as active ingredient.

(43) TABLE-US-00001 TABLE 1 Biofilm quantity after 48 h contact time Dosage of Biofilm DBNPA Abs. at reduction [mg/l] 595 nm [%] 0 0.66 0.2 0.57 16.9 0.6 0.35 60.7 1 0.15 98.8

(44) Table 2 shows the effect of DPNPA dosing to Pseudoxanthomonas taiwanensis biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was stained and quantified by absorbance measurement. Dosage given as active ingredient.

(45) TABLE-US-00002 TABLE 2 Biofilm quantity after 48 h contact time Dosage of Biofilm DBNPA Abs. at reduction, [mg/l] 595 nm [%] 0 1.65 0.2 1.46 12.6 0.6 1.23 27.8 1 0.14 99.9

Example 2 (Reference)

(46) Tables 3 and 4 show effect of a well-known antibiotic Gramicidin against biofilm formation of Meiothermus silvanus and Pseudoxanthomonas taiwanensis. In a synthetic growth medium R2-broth Gramicidin was capable to prevent biofilm formation at clearly lower concentration than in conditions simulating paper or board making process (synthetic paper machine water, high temperature, fibres present, high flow).

(47) The results in Table 3 and 4 demonstrate expected behaviour of a clinical antimicrobial compound with deteriorating performance when exposed to non-clinical conditions.

(48) Table 3 shows the effect of Gramicidin dosing to Meiothermus silvanus biofilms in R2-broth and SPW. Biofilm was stained and quantified by absorbance measurement. Dosage given as active ingredient.

(49) TABLE-US-00003 TABLE 3 Biofilm quantity after 48 h Biofilm quantity after 48 h contact time in R2-broth contact time in SPW Dosage of Biofilm Biofilm Gramicidin Abs. at reduction, Abs. at reduction, [mg/l] 595 nm [%] 595 nm [%] 0 1.60 — 1.36 — 0.2 1.40 13.7 1.33 2.5 1 0.66 64.4 1.41 −4.1 3 0.17 97.9 0.45 74.6 10 0.14 100.0 0.19 95.9

(50) Table 4 shows the effect of Gramicidin dosing to Pseudoxanthomonas taiwanensis biofilms in R2-broth and SPW. Biofilm was stained and quantified by absorbance measurement. Dosage given as active ingredient.

(51) TABLE-US-00004 TABLE 4 Biofilm quantity after 48 h Biofilm quantity after 48 h contact time in R2-broth contact time in SPW Dosage of Biofilm Biofilm Gramicidin Abs. at reduction, Abs at reduction, [mg/l] 595 nm [%] 595 nm [%] 0 2.78 — 2.37 — 3 2.80 −0.8 2.25 5.4 10 2.55 8.7 2.41 −1.8 25 0.19 98.1 2.42 −2.2

Example 3

(52) Tables 5 and 6 demonstrate the ability of Compound C and Compound E to prevent biofilm formation of Meiothermus silvanus and Pseudoxanthomonas taiwanensis. Test conditions are identical to test conditions of Example 1. It was observed that Compound C and Compound E were able to control biofilms at a very low concentration. Already a dosage of 0.2 mg/I active Compound C or Compound E gave over 90% biofilm reduction effect.

(53) Table 5 shows the effect of Compound C dosage to Meiothermus silvanus biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was stained and quantified by absorbance measurement. Compound C dosage is given as active compound.

(54) TABLE-US-00005 TABLE 5 Biofilm quantity after 48 h contact time Dosage of Biofilm Compound C Abs. at reduction [mg/l] 595 nm [%] 0 0.85 0.06 0.64 29.7 0.2 0.15 98.2

(55) Table 6 shows the effect of Compound E dosage to Meiothermus silvanus biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was stained and quantified by absorbance measurement. Compound E dosage is given as active compound.

(56) TABLE-US-00006 TABLE 6 Biofilm quantity after 48 h contact time Dosage of Biofilm Compound E Abs. at reduction [mg/l] 595 nm [%] 0 2.25 0.06 1.43 38.8 0.2 0.14 99.6

(57) Results in Tables 5 and 6 demonstrate that Compound C and Compound E are capable to prevent biofilm formation of dominant industrial biofilm-formers under paper machine conditions at a very low dosage when compared to conventional biocide used in paper industry.

Example 4

(58) Tables 7 and 8 demonstrate the ability of Compound D and Compound F to remove already formed biofilms of Meiothermus silvanus or Pseudoxanthomonas taiwanensis. Test conditions simulated paper making process conditions (synthetic paper machine water, high temperature, fibres present, high flow). Compound D and Compound F were observed to remove already formed biofilms.

(59) Table 7 shows the effect of Compound D dosage to Pseudoxanthomonas taiwanensis biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was pre-grown for 24 h after which Compound D was added in given amount. After 24 hours the biofilm was stained and quantified by absorbance measurement. Compound D dosage is given as active compound.

(60) TABLE-US-00007 TABLE 7 Biofilm quantity after 24 h pre-growth and 24 h contact time Dosage of Biofilm Compound D Abs. at reduction [mg/l] 595 nm [%] 0 2.25 0.2 2.07 8.4 0.6 0.18 97.9

(61) Table 8 shows the effect of Compound F dosage to Meiothermus silvanus biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was pre-grown for 24 h after which Compound F was added in given amount. After 24 hours the biofilm was stained and quantified by absorbance measurement. Compound F dosage is given as active compound.

(62) TABLE-US-00008 TABLE 8 Biofilm quantity after 24 h pre-growth and 24 h contact time Dosage of Biofilm Compound F Abs. at reduction [mg/l] 595 nm [%] 0 1.29 0.2 1.21 6.4 0.6 0.86 37.3

Example 5

(63) Table 9 demonstrates the ability of Compound C to remove already formed biofilms of Pseudoxanthomonas taiwanensis. Test conditions simulated paper making process conditions (synthetic paper machine water, high temperature, fibres present, high flow). Compound C was observed to remove already formed biofilms.

(64) Table 9 shows the effect of Compound C dosage to Pseudoxanthomonas taiwanensis biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was pre-grown for 24 h after which Compound C was added in given amount. After 24 hours the biofilm was stained and quantified by absorbance measurement. Compound C dosage is given as active compound.

(65) TABLE-US-00009 TABLE 9 Biofilm quantity after 24 h pre-growth and 24 h contact time Dosage of Biofilm Compound C Abs. at reduction [mg/l] 595 nm [%] 0 1.05 0.2 0.15 98.5 0.4 0.15 99.0

(66) Even if the invention was described with reference to what at present seems to be the most practical and preferred embodiments, it is appreciated that the invention shall not be limited to the embodiments described above, but the invention is intended to cover also different modifications and equivalent technical solutions within the scope of the enclosed claims.